Carbazole-containing amides, carbamates, and ureas as cryptochrome modulators

ABSTRACT

The subject matter herein is directed to carbazole-containing amide, carbamate, and urea derivatives and pharmaceutically acceptable salts or hydrates thereof of structural formula I wherein the variable R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , A, D, E, G, J, L, M, Q, a, and b are accordingly described. Also provided are pharmaceutical compositions containing the compounds of formula I to treat a Cry-mediated disease or disorder, such as diabetes, complications associated with diabetes, Cushing&#39;s syndrome, NASH, NAFLD, asthma, and COPD.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.61/976,350 filed on Apr. 7, 2014, the entirety of which is hereinincorporated by reference.

TECHNICAL FIELD

The subject matter disclosed herein relates to, inter alia,carbazole-containing amide, carbamate, and urea derivatives,pharmaceutical compositions containing these compounds, methods fortheir use in treating cryptochrome-mediated diseases or disorders, andprocesses for their production. Also provided are methods of diagnosing,detecting, or monitoring the progression of cryptochrome-dependentdiseases in subjects receiving the compounds and compositions disclosedherein.

BACKGROUND

The circadian clock is an intrinsic time-keeping mechanism that controlsthe daily rhythms of many physiological processes, such as sleep/wakebehavior, body temperature, hormone secretion, and metabolism(Takahashi, J. S. et al. Nat. Rev. Genet. 2008, 9, 764; Green, C. B. etal. Cell, 2008, 134, 728; Zhang, E. E. et al. Nat. Rev. Mol. Cell. Biol.2010, 11, 764). Circadian rhythms are generated in a cell-autonomousmanner through transcriptional regulatory networks of clock genes. Inthe core feedback loop, the transcription factors CLOCK and BMAL1activate expression of Period (Per1 and Per2) and Cryptochrome (Cry1 andCry2) genes. After translation and nuclear localization, PER and CRYproteins inhibit the function of CLOCK-BMAL1, resulting in sustainedrhythmic gene expression. Many physiological pathways are under thecontrol of the circadian clock (Panda, S. et al. Cell, 2002, 109, 307),including direct regulation of numerous hepatic processes (Rey, G. etal. PLoS Biol. 2011, 9, e1000595; Bugge, A. et al. Genes Dev. 2012, 26,657).

Circadian desynchrony has been associated with impaired insulinsensitivity (Spiegel, K. et al. J. Appl. Physiol. 2005, 99, 2008;Spiegel, K. et al. Lancet, 1999, 354, 1435), decreased leptin levels andresults in hyperglycemia, hyperinsulinemia and postprandial glucoseresponses comparable to those of a prediabetic state (Scheer, F. A. etal. Proc. Natl. Acad. Sci. USA, 2009, 106, 4453). Several genome-wideassociation studies led to the discovery that Cry2 may be important inthe regulation of mammalian glucose levels (Dupuis, J. et al. Nat.Genet. 2010, 42, 105; Liu, C. et al. PLoS One, 2011, 6, e21464; Barker,A. et al. Diabetes, 2011, 60, 1805).

Glucose concentrations in the blood are highly rhythmic because ofchanges in insulin sensitivity and insulin secretory capacity of theendocrine pancreas (Polonsky, K. S. et al. N. Engl. J. Med. 1988, 318,1231). Clock^(Δ19) mutant mice develop age-dependent hyperglycemia andthese animals also develop susceptibility to diet-induced obesity, haveinappropriately low concentrations of insulin (Turek, F. W. et al.Science, 2005, 308, 1043) and display a steeper drop in blood sugar inresponse to treatment with insulin, indicating that these animals haveenhanced insulin sensitivity, thereby masking their β-cell deficiency(Marcheva, B. et al. Nature, 2010, 466, 627). Liver-specific deletion ofBmal1 in mice results in impaired glucose tolerance and increasedinsulin sensitivity (Lamia, K. A. et al. Proc. Natl. Acad. Sci. USA,2008, 105, 15172). Individuals with type 2 diabetes, and even theirfirst-degree relatives not yet affected with the disease, displayaltered rhythmicity in glucose tolerance (Boden, G. et al. Diabetes,1999, 48, 2182). Also, Per2, Per3, and Cry2 expression is significantlylower in humans with type 2 diabetes versus humans without the disease(Stamenkovich, J. A. et al. Metabolism, 2012, 61, 978). Thegluconeogenic genes phosphoenol pyruvate carboxykinase (Pck1) andglucose 6-phosphatase (G6pc) are controlled by CRY and the Bmal1 generegulator REV-ERB (Zhang, E. E. et al. Nat. Med. 2010, 16, 1152; Lamia,K. A. et al. Nature, 2011, 480, 552; Yin, L. et al. Science, 2007, 318,1786). Gluconeogenesis is tightly controlled by multiple signalingmechanisms and moreover, studies in mice have revealed that modulationof Cry1 and Cry2 can perturb gluconeogenesis and regulate blood sugarlevels (Zhang, E. E. et al. Nat. Med. 2010, 16, 1152).

In a monotherapeutic or combination therapy context, new and establishedoral antidiabetic agents have non-uniform and limited effectiveness.Oral antidiabetic therapies suffer from poor or limited glycemiccontrol, or poor patient compliance due to unacceptable side effects,such as edema, weight gain, or even more serious complications likehypoglycemia. Metformin, a substituted biguanide, can cause diarrhea andgastrointestinal discomfort. Finally, edema, weight gain, and in somecases, hepatotoxicity and cardiotoxicity, have been linked to theadministration of some thiazolidine-2,4-dione antidiabetic agents (e.g.Rosiglitazone and Pioglitazone). Combination therapy using two or moreof the above agents is common, but generally only leads to incrementalimprovements in glycemic control.

Cry1 and Cry2 also interact with the glucocorticoid receptor (GR) toglobally alter the transcriptional response to glucocorticoids (Lamia,K. A. et al. Nature, 2011, 480, 552). Loss of Cry1 and/or Cry2 resultsin glucose intolerance and constitutively high levels of circulatingcorticosterone, suggesting reduced suppression of thehypothalamic-pituitary-adrenal axis coupled with increasedglucocorticoid transactivation in the liver. Genomically, Cry1 and Cry2associate with a glucocorticoid response element in the Pck1 promoter ina hormone-dependent manner, and dexamethasone-induced transcription ofthe Pck1 gene was strikingly increased in cryptochrome-deficient livers.This suggests that the undesirable metabolic side effects ofglucocorticoids (e.g. hyperglycemia, insulin resistance and suppressionof adrenal function) used to suppress inflammation may be alleviated bycombining them with agents that can stabilize Cry1 and/or Cry2.

SUMMARY

The subject matter herein relates to cryptochrome (Cry) modulatingcompounds, pharmaceutical compositions containing the Cry modulatingcompounds and methods of treating Cry-related diseases or disorders,such as, e.g. diabetes, obesity, metabolic syndrome, Cushing's syndromeand glaucoma, by administration of Cry modulating compounds.

In one aspect, the subject matter disclosed herein is directed to acompound of formula I:

or a pharmaceutically acceptable salt or hydrate thereof, wherein:

each of A, D, E, G, J, L, M, and Q is independently N or C;

each of R₁ and R₂, when A, D, E, G, J, L, M, and Q is C, isindependently selected from H, halo, cyano, nitro, —CF₃, —CHF₂, —CH₂F,trifluoromethoxy, azido, hydroxyl, (C₁-C₆)alkoxy, (C₁-C₆)alkyl,(C₂-C₆)alkenyl, (C₂-C₆)alkynyl, —(C═O)—R₈, —(C═O)—O—R₈, —O—(C═O)—R₈,—NR₈(C═O)—R₁₀, —(C═O)—NR₈R₉, —NR₈R₉, —NR₈OR₉, —S(O)_(c)NR₈R₉,—S(O)_(d)(C₁-C₈)alkyl, —O—SO₂—R₈, NR₈—S(O)_(c),—(CR₈R₉)_(d)(3-10)-membered cycloalkyl, —(CR₈R₉)_(e)(C₆-C₁₀)aryl,—(CR₈R₉)_(e)(4-10)-membered heterocyclyl,—(CR₈R₉)_(f)(C═O)(CR₈R₉)_(e)(C₆-C₁₀)aryl,—(CR₈R₉)_(f)(C═O)(CR₈R₉)_(e)(4-10)-membered heterocyclyl,—(CR₈R₉)_(e)O(CR₈R₉)_(f)(C₆-C₁₀)aryl,—(CR₈R₉)_(e)O(CR₈R₉)_(f)(4-10)-membered heterocyclyl,—(CR₈R₉)_(f)S(O)_(d)(CR₈R₉)_(e)(C₆-C₁₀)aryl, and—(CR₈R₉)_(f)S(O)_(d)(CR₈R₉)_(e)(4-10)-membered heterocyclyl;

each of R₃ and R₅ is independently selected from H, cyano, —CF₃, —CHF₂,—CH₂F, (C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, —(C═O)—R₈,—(C═O)—O—R₈, —(C═O)—NR₈R₉, —S(O)_(c)NR₈R₉, —S(O)_(d)(C₁-C₈)alkyl,—(CR₈R₉)_(d)(3-10)-membered cycloalkyl, —(CR₈R₉)_(e)(C₆-C₁₀)aryl,—(CR₈R₉)_(e)(4-10)-membered heterocyclyl,—(CR₈R₉)_(f)(C═O)(CR₈R₉)_(e)(C₆-C₁₀)aryl,—(CR₈R₉)_(f)(C═O)(CR₈R₉)_(e)(4-10)-membered heterocyclyl,—(CR₈R₉)_(e)O(CR₈R₉)_(f)(C₆-C₁₀)aryl,—(CR₈R₉)_(e)O(CR₈R₉)_(f)(4-10)-membered heterocyclyl,—(CR₈R₉)_(f)S(O)_(d)(CR₈R₉)_(e)(C₆-C₁₀)aryl, and—(CR₈R₉)_(f)S(O)_(d)(CR₈R₉)_(e)(4-10)-membered heterocyclyl;

wherein each of the R₃ groups are optionally linked to each other as a4-12 membered mono- or bicyclic ring;

wherein each of the R₅ groups are optionally linked to each other as a4-12 membered mono- or bicyclic ring;

R₄ is H, —CF₃, —CHF₂, —CH₂F, (C₁-C₆)alkyl, (C₂-C₆)alkenyl,(C₂-C₆)alkynyl, —(C═O)—R₈, —(C═O)—O—R₈, —(C═O)—NR₈R₉,—(CR₈R₉)_(d)(3-10)-membered cycloalkyl, —(CR₈R₉)_(e)(C₆-C₁₀)aryl,—(CR₈R₉)_(e)(4-10)-membered heterocyclyl,—(CR₈R₉)_(f)(C═O)(CR₈R₉)_(e)(C₆-C₁₀)aryl,—(CR₈R₉)_(f)(C═O)(CR₈R₉)_(e)(4-10)-membered heterocyclyl,—(CR₈R₉)_(e)O(CR₈R₉)_(f)(C₆-C₁₀)aryl,—(CR₈R₉)_(e)O(CR₈R₉)_(f)(4-10)-membered heterocyclyl,—CR₈R₉)_(f)S(O)_(d)(CR₈R₉)_(e)(C₆-C₁₀)aryl, and—(CR₈R₉)_(f)S(O)_(d)(CR₈R₉)_(e)(4-10)-membered heterocyclyl;

wherein R₆ and R₇ are linked to each other as a 4-12 membered mono- orbicyclic ring;

each of R₈, R₉ and R₁₀ are independently selected from H, (C₁-C₆)alkyl,—(CR₁₁R₁₂)_(e)(3-10)-membered cycloalkyl, —(CR₁₁R₁₂)_(g)(C₆-C₁₀)aryl,and —(CR₁₁R₁₂)_(g)(4-10)-membered heterocyclyl;

any carbon atoms of the (C₁-C₆)alkyl, the (3-10)-membered cycloalkyl,the (C₆-C₁₀)aryl and the (4-10)-membered heterocyclyl of the foregoingR₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, andR₁₆ are independently optionally substituted with 1 to 3 R₁₄substituents each independently selected from halo, cyano, nitro, —CF₃,—CHF₂, —CH₂F, trifluoromethoxy, azido, hydroxyl, —O—R₁₅, (C₁-C₆)alkoxy,(C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, —(C═O)—R₁₁, —(C═O)—R₁₅,—(C═O)—O—R₁₁, —(C═O)—O—R₁₅, —O—(C═O)—R₁₁, —O—(C═O)—R₁₅, —NR11(C═O)—R₁₃,—(C═O)—NR₁₁R₁₂, —(C═O)—NR₁₁R₁₅, —NR₁₁R₁₂, —NR₁₁R₁₅, —NR₁₁OR₁₂,—NR₁₁OR₁₅, —S(O)_(c)NR₁₁R₁₂, —S(O)_(c)NR₁₁R₁₅, —S(O)_(d)(C₁-C₆)alkyl,—S(O)_(d)R₁₅, —O—SO₂—R₁₁, —O—SO₂—R₁₅, —NR₁₁—S(O)_(c), —NR₁₅—S(O)_(c),—(CR₁₁R₁₂)_(e)(3-10)-membered cycloalkyl, —(CR₁₁R₁₂)_(e)(C₆-C₁₀)aryl,—(CR₁₁R₁₂)_(e)(4-10)-membered heterocyclyl,—(CR₁₁R₁₂)_(f)(C═O)(CR₁₁R₁₂)_(e)(C₆-C₁₀)aryl,—(CR₁₁R₁₂)_(f)(C═O)(CR₁₁R₁₂)_(e)(4-10)-membered heterocyclyl,—(CR₁₁R₁₂)_(e)O(CR₁₁R₁₂)_(f)(C₆-C₁₀)aryl,—(CR₁₁R₁₂)_(e)O(CR₁₁R₁₂)_(f)(4-10)-membered heterocyclyl,—(CR₁₁R₁₂)_(f)S(O)_(d)(CR₁₁R₁₂)_(e)(C₆-C₁₀)aryl, and—(CR₁₁R₁₂)_(f)S(O)_(d)(CR₁₁R₁₂)_(e)(4-10)-membered heterocyclyl;

any carbon atoms of the (C₁-C₆)alkyl, the (3-10)-membered cycloalkyl,the (C₆-C₁₀)aryl and the (4-10)-membered heterocyclyl of the foregoingR₁₄ are independently optionally substituted with 1 to 3 R₁₆substituents each independently selected from halo, cyano, nitro, —CF₃,—CHF₂, —CH₂F, trifluoromethoxy, azido, (CH₂)_(e)OH, (C₁-C₆)alkoxy,(C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, —(C═O)—R₁₁, —(C═O)—R₁₅,—(C═O)—O—R₁₁, —(C═O)—O—R₁₅, —O—(C═O)—R₁₁, —O—(C═O)—R₁₅, —NR₁₁(C═O)—R₁₃,—(C═O)—NR₁₁R₁₂, —NR₁₁R₁₂, and —NR₁₁R₁₅;

any nitrogen atoms of the (4-10)-membered heterocyclyl of the foregoingR₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₄, and R₁₅ are independentlyoptionally substituted with (C₁-C₆)alkyl, (C₂-C₆)alkenyl,(C₂-C₆)alkynyl, —(C═O)—R₁₁, —(C═O)—O—R₁₁, —(C═O)—NR₁₁R₁₂,—(CR₁₁R₁₂)_(e)(3-10)- membered cycloalkyl, —(CR₁₁R₁₂)_(e)(C₆-C₁₀)aryl,—(CR₁₁R₁₂)_(e)(4-10)-membered heterocyclyl,—(CR₁₁R₁₂)_(f)(C═O)(CR₁₁R₁₂)_(e)(C₆-C₁₀)aryl, or—(CR₁₁R₁₂)_(f)(C═O)(CR₁₁R₁₂)_(e)(4-10)-membered heterocyclyl;

each R₁₁, R₁₂, and R₁₃ are independently H or (C₁-C₆)alkyl;

R₁₅ is —(CR₁₁R₁₂)_(e)(3-10)-membered cycloalkyl,—(CR₁₁R₁₂)_(e)(C₆-C₁₀)aryl, or —(CR₁₁R₁₂)_(e)(4-10)-memberedheterocyclyl;

a and b are each independently 1, 2, 3, or 4;

c is 1 or 2;

d is 0, 1, or 2; and

e, f, and g are each independently 0, 1, 2, 3, 4, or 5.

In some embodiments, each of A, D, E, G, J, L, M, and Q are C; each ofR₁ and R₂ is independently selected from H or halo; R₄ is H or(C₁-C₆)alkyl, R₃ and R₅ are H; R₆ and R₇ are linked to each other as a4-12 membered mono- or bicyclic amide ring; R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃,R₁₄, R₁₅, R₁₆, a, b, c, d, e, and f are as defined herein.

In other embodiments, each of A, D, E, G, J, L, M, and Q are C; each ofR₁ and R₂ is independently selected from H or halo; R₄ is H or(C₁-C₆)alkyl, R₃ and R₅ are H; R₆ and R₇ are linked to each other as a4-12 membered mono- or bicyclic urea ring; R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃,R₁₄, R₁₅, R₁₆, a, b, c, d, e, and f are as defined herein.

In some embodiments, the compound of formula I is a single enantiomerbearing an (R)-configuration at C-3, wherein each of A, D, E, G, J, L,M, and Q are C; each of R₁ and R₂ is independently selected from H orhalo; R₄ is H or (C₁-C₆)alkyl, R₃ and R₅ are H; R₆ and R₇ are linked toeach other as a 4-12 membered mono- or bicyclic amide ring; R₈, R₉, R₁₀,R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, a, b, c, d, e, and f are as definedherein.

In other embodiments of the subject matter disclosed herein, thecompound of formula I is a single enantiomer bearing an(R)-configuration at C-3, wherein each of A, D, E, G, J, L, M, and Q areC; each of R₁ and R₂ is independently selected from H or halo; R₄ is Hor (C₁-C₆)alkyl, R₃ and R₅ are H; R₆ and R₇ are linked to each other asa 4-12 membered mono- or bicyclic urea ring; R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃,R₁₄, R₁₅, R₁₆, a, b, c, d, e, and f are as defined herein.

Other embodiments of the subject matter described herein are compoundsselected from the group consisting of:

-   1-(3-(3,6-difluoro-9H-carbazol-9-yl)-2-hydroxypropyl)-3-fluoropyrrolidin-2-one;-   2-(3-(3,6-difluoro-9H-carbazol-9-yl)-2-hydroxypropyl)-2-azabicyclo[2.2.1]heptan-3-one;-   1-(3-(3,6-difluoro-9H-carbazol-9-yl)-2-hydroxypropyl)imidazolidin-2-one;-   (1R,4S)-2-((R)-3-(3,6-difluoro-9H-carbazol-9-yl)-2-hydroxypropyl)-2-azabicyclo[2.2.1]heptan-3-one;-   (R)-1-(3-(3,6-difluoro-9H-carbazol-9-yl)-2-hydroxypropyl)imidazolidin-2-one;-   (R)-1-((R)-3-(3,6-difluoro-9H-carbazol-9-yl)-2-hydroxypropyl)-3-fluoropyrrolidin-2-one;-   (S)-1-((S)-3-(9H-carbazol-9-yl)-2-hydroxy-2-methylpropyl)-3-fluoropyrrolidin-2-one;-   (R)-1-((R)-3-(9H-carbazol-9-yl)-2-hydroxypropyl)-4-methylimidazolidin-2-one;    or a pharmaceutically acceptable salt or hydrate thereof.

In another aspect, the compounds described herein modulate Cry1 or Cry2.Modulation of Cry1 or Cry2 includes any one of the following: binding toCry1 or Cry2; inhibiting modification of Cry1 or Cry2; altering Cry1 orCry2 localization; increasing or decreasing Cry1 or Cry2 stabilization;increasing or decreasing the binding between Cry1 or Cry2 to a target;increasing or decreasing Cry1 or Cry2 activity; and increasing ordecreasing activity of a Cry1 or Cry2 target. Targets of Cry1 and/orCry2 include, but are not limited to, Per1, Per2, glucocorticoidreceptor (GR), CLOCK, BMAL1, or a CLOCK-BMAL1 promoter sequence.

In another aspect, the subject matter described herein provides apharmaceutical composition comprising a compound of formula I, or apharmaceutically acceptable salt or hydrate thereof, and apharmaceutically acceptable carrier, adjuvant, or diluent. In someembodiments, the pharmaceutical composition further comprises one ormore additional therapeutic agents. Examples of additional therapeuticagents include, but are not limited to, DPP-IV inhibitors such assitagliptin, alogliptin, vildagliptin, saxagliptin and linagliptin;GLP-1 agonists such as exenatide, liraglutide and albiglutide; SGLT2inhibitors such as canagliflozin, ertugliflozin, and dapagliflozin);metformin; and sulfonylureas such as glyburide. Other examples ofadditional therapeutic agents includes Signifor®, ketoconazole,metyrapone, mitotane, etomidate, Korlym®, epidermal growth factorinhibitors, the aldosterone synthase/11β-hydroxylase inhibitor LCI699,and kevoketoconazole (COR-003).

In other aspects, a method of treating a Cry-mediated disease ordisorder in a subject is provided, by administering to the subject atherapeutically effective amount of the pharmaceutical compositiondescribed herein. In a further aspect, the present invention provides amethod for alleviating a symptom of a Cry-mediated disease or disorderin a subject, by administering to the subject a therapeuticallyeffective amount of the pharmaceutical composition described herein. Thedisease or disorder may be selected from the group consisting ofdiabetes, diabetic complications, such as diabetic neuropathy, diabeticretinopathy, diabetic nephropathy, cataract formation, glaucoma,diabetic angiopathy, atherosclerosis; nonalcoholic steatohepatitis(NASH); non-alcoholic fatty liver disease (NAFLD); asthma; chronicobstructive pulmonary disease (COPD); metabolic syndrome; insulinresistance syndrome; obesity; glaucoma; Cushing's syndrome; psychoticdepression; Alzheimer's disease; neuropathic pain; drug abuse;osteoporosis; cancer; macular degeneration; and myopathy.

Any of the methods described herein can also involve the administrationof one or more additional therapeutic agents to the subject. Examples ofadditional therapeutic agents include, but are not limited to, DPP-IVinhibitors such as sitagliptin, alogliptin, vildagliptin, saxagliptinand linagliptin; GLP-1 agonists such as exenatide, liraglutide andalbiglutide; SGLT2 inhibitors such as canagliflozin, ertugliflozin, anddapagliflozin); metformin; sulfonylureas, such as glyburide; Signifor®;ketoconazole; metyrapone; mitotane; etomidate; Korlym®; epidermal growthfactor inhibitors; the aldosterone synthase/11β-hydroxylase inhibitorLCI699; and kevoketoconazole (COR-003)

In another aspect, a method of monitoring progression or prognosis of aCry-mediated disease or disorder in a subject is provided, involvingmeasuring an effective amount of one or more cryptochromes orcryptochrome-regulated genes in a first sample from the subject at afirst period of time; measuring an effective amount of one or morecryptochromes or cryptochrome-regulated genes in a second sample fromthe subject at a second period of time; and comparing the amount of theone or more cryptochromes or cryptochrome-regulated genes detected inthe first sample to the amount of the one or more cryptochromes orcryptochrome-regulated genes detected in the second sample, or to areference value. Examples of cryptochrome-regulated genes include genesthat contain an E-box sequence in their promoter. Such genes include,but are not limited to Dbp, Rev-erb alpha, Rev-erb beta, Ror alpha, Rorbeta, Ror gamma, Per1, Per2, Per3, Cry1, Cry2, Pck1, G6Pc, Avp, Vip,Cck, SP (substance P), AA-Nat, PK2 (Prokinectin 2), c-Myc, MyoD andNampt.

In some embodiments, the monitoring comprises evaluating changes in therisk of developing the Cry-mediated disease or disorder in the subject.

The optimal time for dosing in humans is expected to be the evening,corresponding to the peak of human Cry expression and the end of theactive (daytime) period.

The subject may comprise one who has been previously treated for theCry-mediated disease or disorder, one who has not been previouslytreated for the Cry-mediated disease or disorder, or one who has notbeen previously diagnosed with the Cry-mediated disease or disorder. Thesample can be whole blood, serum, plasma, blood cells, endothelialcells, tissue biopsies, lymphatic fluid, ascites fluid, interstitialfluid, bone marrow, cerebrospinal fluid (CSF), seminal fluid, saliva,mucous, sputum, sweat, or urine.

In some embodiments, the first sample is taken from the subject prior tobeing treated for the Cry-mediated disease or disorder and the secondsample is taken from the subject after being treated for theCry-mediated disease or disorder. In other embodiments, the subject istreated with the pharmaceutical composition containing the compounds offormula I disclosed herein. In certain embodiments, the monitoringfurther comprises selecting a treatment for the subject and/ormonitoring the effectiveness of a treatment for the Cry-mediated diseaseor disorder, wherein the treatment for the Cry-mediated disease ordisorder comprises surgical intervention, administration of thepharmaceutical composition as defined herein alone or in combinationwith one or more additional therapeutic agents, surgical interventionfollowing or preceded by administration of the pharmaceuticalcomposition provided herein or in combination with one or moreadditional therapeutic agents, or taking no further action.

In other embodiments, the reference value comprises an index value, avalue derived from one or more Cry-mediated disease or disorder riskprediction algorithms, a value derived from a subject not having aCry-mediated disease or disorder, or a value derived from a subjectdiagnosed with a Cry-mediated disease or disorder. In some embodiments,the measuring comprises detecting the presence or absence of the one ormore cryptochromes, quantifying the amount of the one or morecryptochromes, qualifying the type of the one or more cryptochromes, andassessing the ability of one or more cryptochromes to bind to a target.The target may be Per1, Per2, or a CLOCK-BMAL1 promoter sequence.

As disclosed herein, the Cry-mediated disease or disorder may beselected from the group consisting of diabetes, diabetic complicationssuch as diabetic neuropathy, diabetic retinopathy, diabetic nephropathy,cataract formation, glaucoma, diabetic angiopathy, atherosclerosis;nonalcoholic steatohepatitis (NASH); non-alcoholic fatty liver disease(NAFLD); asthma; chronic obstructive pulmonary disease (COPD); metabolicsyndrome; insulin resistance syndrome; obesity; glaucoma; Cushing'ssyndrome; psychotic depression; Alzheimer's disease; neuropathic pain;drug abuse; osteoporosis; cancer; macular degeneration; and myopathy.

In one embodiment, in the compounds of formula I disclosed herein, A, D,E, G, J, L, M, and Q are carbon. In another embodiment, in the compoundsof formula I, R₁ and R₂ are hydrogen. In still other embodiments, in thecompounds of formula I, R₁ and R₂ are fluorine and a and b are 1. Infurther embodiments, in the compounds of formula I, R₃ and R₅ arehydrogen. In another embodiment, in the compounds of formula I, R₃, R₄,and R₅ are hydrogen. In some embodiments, in the compounds of formula I,R₆ and R₇ are linked to form an optionally substituted monocyclic ring.

In other embodiments, in the compounds of formula I, R₆ and R₇ arelinked to form an optionally substituted fused bicyclic ring, anoptionally substituted bridged bicyclic ring, an optionally substitutedspiro bicyclic ring, an optionally substituted pyrrolidinone ring, anoptionally substituted imidazolidinone ring, an optionally substitutedpiperidinone ring, and/or an optionally substituted pyrimidinone ring.Likewise, in any of these embodiments, the ring formed by R₆ and R₇ canbe substituted exclusively with fluorine, methyl groups, ethyl groups,isopropyl groups, C₃-6 cycloalkanes, or phenyl groups.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice of the present invention, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are expressly incorporated byreference in their entirety. In cases of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples described herein are illustrative onlyand are not intended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-H are a series of graphs showing core clock gene expression inmice after administration of Compound 72. mRNA expression of core clockgenes Per2 (A and B), Bmal1 (C and D), Cry1 (E and F), and Cry2 (G andH) was measured in six hour intervals over 24 hours in the livers ofC57Bl/6J DIO (A, C, E, G) or Balb/c (B, D, F, H) mice treated withvehicle (H₂O) or Compound 72. Transcript levels were determined byRT-qPCR and compared to vehicle at ZT8 with the dark period shaded. ThemRNA levels from Compound 72-treated for each time point was compared tovehicle by T-test: * <0.05, ** <0.01, *** <0.001, **** <0.0001.

FIGS. 2A-D are a series of graphs showing gluconeogenic gene expressionin mice after administration of Compound 72. mRNA expression ofgluconeogenic genes Pck1 (PEPCK; A and B), G6Pc (Glucose 6-phosphatasecatalytic subunit; C and D) at six hour intervals over 24 hours in thelivers of C57Bl/6J DIO (A and C) or Balb/c (B and D) mice treated withvehicle (H₂O) or Compound 72. Transcript levels were determined byRT-qPCR and compared to vehicle at ZT8 with the dark period shaded. ThemRNA levels from Compound 72-treated for each time point was compared tovehicle by T-test: * <0.05, ** <0.01, *** <0.001, **** <0.0001.

FIGS. 3A-C are a series of graphs showing core clock gene expression inthe livers of ICR mice after administration of Compound 72, Compound 48,Compound 9, or Compound 57. mRNA expression of core clock genes Per2(A), Bmal1 (B), and Cry2 (C) was measured in the livers of ICR micetreated for 4 days BID with Compound 72, Compound 48, Compound 9,Compound 57 or vehicle. Levels of the mRNAs were determined by RT-qPCRon samples taken at ZT6 after the final ZT0 dose. The mRNA levels fromCompound 72-treated for each time point was compared to vehicle byT-test: * <0.05, ** <0.01, *** <0.001, **** <0.0001.

FIG. 4 is a graph showing Dbp gene expression after three daily doses ofCompound 72 at the peak of Cry1 expression. Expression of Dbp mRNA wasmeasured at ZT7.5 in whole blood from db/db mice after three daily dosesof 100 mg/kg Compound 72. Transcript levels were determined by RT-qPCRand compared to blood from vehicle (10% kolliphor)-treated mice atZT7.5. The mRNA levels from each compound treatment was compared tovehicle by T-test (***; p≦0.001).

FIGS. 5A-D are a series of graphs showing core clock gene expressionafter a single dose of Compound 72 at the peak or nadir of Cry1expression. mRNA expression of core clock genes Per2 (A), Bmal1 (B),Cry1 (C) and Cry2 (D) was measured at ZT7.5 (Peak of Cry1 expression) orZT17.5 (nadir of Cry1 expression) in liver from C57Bl/6J DIO mice aftera single dose of 100 mg/kg Compound 72. Transcript levels weredetermined by RT-qPCR and compared to liver from vehicle (10%kolliphor)-treated. The mRNA levels from Compound 72-treated for eachtime point was compared to vehicle by T-test: * <0.05, ** <0.01, ***<0.001, **** <0.0001.

FIG. 6 is a series of graphs showing the effect of Compound 72 on oralglucose tolerance test (OGTT) in db/db mice. Compound 72 (50 mg/kg, PO)or 10% kolliphor (control) was administered as a single dose either atthe peak (ZT0) (A) or nadir (ZT10) (B) of Cry1 and Bmal1 geneexpression.

FIG. 7 is a graph showing the effect of Compound 72 on glucose areaunder the curve (AUC) in db/db mice. Compound 72 (50 mg/kg, PO) or 10%kolliphor (control) was administered as a single dose at the peak (ZT0)of Cry1 and Bmal1 gene expression.

FIGS. 8A-C are a series of graphs showing the effect of Compound 72administered for 7 days on glucose metabolism in db/db mice. Compound 72(50 mg/kg, PO) or 10% kolliphor (control) was administered for 7 days.A) Fasting blood glucose levels; B) oral glucose tolerance test (OGTT);C) glucose AUC.

FIGS. 9A-B are a series of graphs showing the effect of Compound 72administered for 7 days on insulin levels in db/db mice. Compound 72 (50mg/kg, PO) or 10% kolliphor (control) was administered. A) Insulinlevels before (at 0 h) and after glucose load (at 2 h); B) Homeostaticmodel assessment estimated insulin resistance (HOMA-IR).

FIG. 10 is a graph showing the compound levels of Compound 72 measuredin plasma and liver around 8 hours after administration of the last dose(50 mg/kg, PO). The EC₅₀ concentration for Compound 72 in the Per2 assayis designated on the graph by the dashed line.

FIGS. 11A-C are a series of graphs showing the effect of increasingdosages of Compound 72 (10 mg/kg, 50 mg/kg, and 100 mg/kg) on glucosemetabolism in db/db mice. 10% kolliphor was used as vehicle control. A)Fasting blood glucose levels; B) OGTT; C) glucose AUC.

FIGS. 12A-B are a series of graphs showing the effect of increasingdosages of Compound 72 (10 mg/kg, 50 mg/kg, and 100 mg/kg) on insulinlevels in db/db mice. 10% kolliphor was used as vehicle control. A)Insulin levels before (at 0 h) and after glucose load (at 2 h); B)Homeostatic model assessment estimated insulin resistance (HOMA-IR).

FIG. 13 is a graph showing the compound levels of Compound 72 measuredin plasma and liver around 8 hours after administration of the last doseat increasing dosages (10 mg/kg, 50 mg/kg, and 100 mg/kg). The EC₅₀concentration for Compound 72 in the Per2 assay is designated on thegraph by the dashed line.

FIGS. 14A-C are a series of graphs showing the effect of increasingdosages of Compound 9 (30 mg/kg, 100 mg/kg, and 300 mg/kg) on glucosemetabolism in db/db mice. 10% kolliphor was used as control. A) Fastingblood glucose levels; B) OGTT; C) glucose AUC.

FIGS. 15A-B are a series of graphs showing the effect of varying dosagesof Compound 9 (30 mg/kg, 100 mg/kg, and 300 mg/kg) on insulin levels indb/db mice. 10% kolliphor was used as control. A) Insulin levels before(at 0 h) and after glucose load (at 2 h); B) Homeostatic modelassessment estimated insulin resistance (HOMA-IR).

FIG. 16 is a graph showing the compound levels of Compound 9 in plasmaand liver around 8 hours after administration of the last dose atincreasing dosages (30 mg/kg, 100 mg/kg, and 300 mg/kg). The EC₅₀concentration for Compound 9 in the Per2 assay is designated on thegraph by the dashed line.

FIGS. 17A-C are a graph showing the effect of Compound 72 on glucosemetabolism in C57/Bl6J DIO mice. Compound 72 (100 mg/kg, PO), 10%kolliphor (control), or rosiglitazone (30 mg/kg) was administered for 7days. A) Fasting blood glucose levels; B) OGTT; C) glucose AUC.

FIGS. 18A-B are a series of graphs showing the effect of Compound 72 oninsulin levels in C57/Bl6J DIO mice. Compound 72 (100 mg/kg, PO), 10%kolliphor (control), or rosiglitazone (30 mg/kg) was administered for 7days. A) Insulin levels before (at 0 h) and after glucose load (at 2 h);B) Homeostatic model assessment estimated insulin resistance (HOMA-IR).

FIG. 19 is a series of graphs showing the effect of Compound 72 on a ratmodel of cortisone-induced insulin resistance. Cortisone (30 mg/kg, SC)was administered with either vehicle, Compound 72 (50 mg/kg, PO) ormifepristone (30 mg/kg, PO) for 7 days. (A) Fasted plasma glucose levelsand (B) fasted plasma insulin levels.

FIG. 20 is a graph showing the effect of Compound 72 (50 mg/kg, PO),administered for 7 days on HOMA-IR in a rat model of cortisone-inducedinsulin resistance. Cortisone (30 mg/kg, SC) was administered witheither vehicle, Compound 72 (50 mg/kg, PO) or mifepristone (30 mg/kg,PO) for 7 days.

FIG. 21 is a graph showing the effect of Compound 72 on the in vitrothermal stability of the CRY1 FAD-binding domain. Treatment of purifiedCRY1 FAD-binding domain with Compound 72 caused a dose-dependentincrease in the protein melting temperature, as determined by adifferential scanning fluorimetry (‘thermal shift’) assay.

FIGS. 22A-C are a series of graphs showing the effect of increasingdosages of Compound 72 (10 mg/kg, 30 mg/kg, and 100 mg/kg) on glucosemetabolism in DIO mice. Compound 72 (100 mg/kg, PO), 10% kolliphor(control), or Rosiglitazone (30 mg/kg) was administered for 7 days. A)Fasting blood glucose levels; B) OGTT; C) glucose AUC.

DETAILED DESCRIPTION

Each of the applications and patents cited in this text, as well as eachdocument or reference cited in each of the applications and patents(including during the prosecution of each issued patent; “applicationcited documents”), and each of the U.S. and foreign applications orpatents corresponding to and/or claiming priority from any of theseapplications and patents, and each of the documents cited or referencedin each of the application cited documents, are hereby expresslyincorporated herein by reference. More generally, documents orreferences are cited in this text, either in a Reference List before theclaims, or in the text itself; and, each of these documents orreferences (“herein-cited references”), as well as each document orreference cited in each of the herein-cited references (including anymanufacturer's specifications, instructions, etc.), is hereby expresslyincorporated herein by reference. Documents incorporated by referenceinto this text may be employed in the practice of the invention. Thefeatures, structures, or characteristics described throughout thisspecification may be combined in any suitable manner in one or moreembodiments. For example, the usage of the phrases “exemplaryembodiments,” “example embodiments,” “some embodiments,” or othersimilar language, throughout this specification refers to the fact thata particular feature, structure, or characteristic described inconnection with an embodiment may be included in at least one embodimentdescribed herein. Thus, appearances of the phrases “exemplaryembodiments,” “example embodiments,” “in some embodiments,” “in otherembodiments,” or other similar language, throughout this specificationdo not necessarily all refer to the same group of embodiments, and thedescribed features, structures, or characteristics can be combined inany suitable manner in one or more embodiments.

To facilitate the understanding of this disclosure, a number of termsare defined below. Terms defined herein have meanings as commonlyunderstood by a person of ordinary skill in the areas relevant to thesubject matter described herein. Terms such as “a”, “an” and “the” arenot intended to refer to only a singular entity, but include the generalclass of which a specific example may be used for illustration. Theterminology herein is used to describe specific embodiments of thesubject matter described herein, but their usage does not delimit thesubject matter, except as outlined in the claims.

As used herein, the terms “comprising”, “including”, or “having” areused in their open, non-limiting sense.

The term “halo”, as used herein, unless otherwise indicated, meansfluoro, chloro, bromo, or iodo.

The term “alkyl”, as used herein, unless otherwise indicated, includessaturated monovalent hydrocarbon radicals having straight or branchedmoieties.

The term “alkenyl”, as used herein, represents monovalent straight orbranched chain groups of, unless otherwise specified, from 2 to 6carbons containing one or more carbon-carbon double bonds and isexemplified by ethenyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl,1-butenyl, 2-butenyl, and the like.

The term “alkynyl”, as used herein, represents monovalent straight orbranched chain groups of from two to six carbon atoms containing acarbon-carbon triple bond and is exemplified by ethynyl, 1-propynyl, andthe like.

The term “alkoxy”, as used herein, unless otherwise indicated, includesO-alkyl groups wherein alkyl is as defined above.

The term “Me” means methyl, and “Et” means ethyl.

The term “cycloalkyl”, as used herein, unless otherwise indicated,refers to a non-aromatic, saturated or partially saturated, monocyclicor fused, spiro or unfused bicyclic or tricyclic hydrocarbon referred toherein containing a total of from 3 to 10 carbon atoms. Illustrativeexamples of cycloalkyl are derived from, but not limited to, thefollowing:

The term “aryl”, as used herein, unless otherwise indicated, includes anorganic radical derived from an aromatic hydrocarbon by removal of onehydrogen, such as phenyl or naphthyl.

The term “(4-12)-membered heterocyclyl”, as used herein, unlessotherwise indicated, includes aromatic and non-aromatic heterocyclicgroups containing one to four heteroatoms each selected from O, S, andN, wherein each heterocyclic group has from 4-12 atoms, in its ringsystem, and with the proviso that the ring of said group does notcontain two adjacent O or S atoms. Non-aromatic heterocyclic groupsinclude groups having only 3 atoms in their ring system, but aromaticheterocyclic groups must have at least 5 atoms in their ring system. Theheterocyclic groups include benzo-fused ring systems. An example of a 3membered heterocyclic group is aziridine, an example of a 4 memberedring heterocyclic group is azetidinyl (derived from azetidine). Anexample of a 5 membered heterocyclic group is thiazolyl, an example of a7 membered ring is azepinyl, and an example of a 10 memberedheterocyclic group is quinolinyl. Example of non-aromatic heterocyclicgroups are pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl,tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl,tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino,thioxanyl, piperazinyl, azetidinyl, oxetanyl, thietanyl,homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl,thiazepinyl, 1,2,3,6-tetrahydropyridinyl, 2-pyrrolinyl, 3-pyrrolinyl,indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl,pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl,dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazolidinyl,3-azabicyclo[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptanyl, 3H-indolyl andquinolizinyl. Examples of aromatic heterocyclic groups are pyridinyl,imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl,furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl,quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl,cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl,traizinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl,furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl,benzoxazolyl, quinazolinyl, quinoxalinyl, naphthridinyl, andfuropyridinyl. The foregoing groups, as derived from the lists above,may be C-attached or N-attached where such is possible. For instance, agroup derived from pyrrole may be pyrrol-1-yl (N-attached) orpyrrol-3-yl (C-attached). Further, a group derived from imidazole may beimidazole-1-yl (N-attached) or imidazole-3-yl (C-attached). The 4-12membered heterocyclic may be optionally substituted on any ring carbon,sulfur, or nitrogen atom(s) by one or two oxo, per ring. An example of aheterocyclic group wherein 2 ring atoms are substituted with oxomoieties is 1,1-dioxo-thiomorpholinyl. Other illustrative example of4-12 membered heterocyclic are derived from, but not limited to, thefollowing:

The term “substituted”, as used herein, means that any one or morehydrogen atoms on the designated atom is replaced with a selection fromthe indicated groups, provided that the designated atom's normal valencyis not exceeded, and that the substitution results in a stable compound.When a substituent is keto (i.e., ═O), then 2 hydrogen atoms on the atomare replaced. Keto substituents are not present on aromatic moieties.Ring double bonds, as used herein, are double bonds that are formedbetween two adjacent ring atoms (e.g., C═C, C═N or N═N). Non-limitingexamples of such groups include, without limitation, H, CH₃, NO₂,SO₂N(CH₃)₂, SO₂N((CH₃)SO₂), COOH, COOCH₃, CO(N(CH₃)), alkyl, alkenyl,alkynyl, aryl, aralkyl, cycloalkyl, heterocyclyl, alkylaryl, heteroaryl,heterocycloalkyl, alkoxy (i.e., methoxy, ethoxy, etc), alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, alkylaminocarbonyl, aralkylaminocarbonyl,alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl,alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl,trifluoromethyl, pentafluoroethyl, halogen (i.e., chloro, fluoro, bromo,iodo), cyano, thio, amido, ether, ester, hydroxyl, hydroxyalkyl,saturated or unsaturated fatty acids, azido, phosphonamido, sulfonamido,lactam, phosphate, phosphonato, phosphinato, amino (includingalkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, guanidino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, cyano, azido, etc.

The subject matter disclosed herein provides carbazole-containingsulfonamide compounds that modulate one or more cryptochrome molecules.These compounds have the general structure set forth in formula I:

or a pharmaceutically acceptable salt or hydrate thereof, wherein

each of A, D, E, G, J, L, M, and Q is independently N or C;

each of R₁ and R₂, when A, D, E, G, J, L, M, and Q is C, isindependently selected from H, halo, cyano, nitro, —CF₃, —CHF₂, —CH₂F,trifluoromethoxy, azido, hydroxyl, (C₁-C₆)alkoxy, (C₁-C₆)alkyl,(C₂-C₆)alkenyl, (C₂-C₆)alkynyl, —(C═O)—R₈, —(C═O)—O—R₈, —O—(C═O)—R₈,—NR₈(C═O)—R₁₀, —(C═O)—NR₈R₉, —NR₈R₉, —NR₈OR₉, —S(O)_(c)NR₈R₉,—S(O)_(d)(C₁-C₈)alkyl, —O—SO₂—R₈, NR₈—S(O)_(c),—(CR₈R₉)_(d)(3-10)-membered cycloalkyl, —(CR₈R₉)_(e)(C₆-C₁₀)aryl,—(CR₈R₉)_(e)(4-10)-membered heterocyclyl,—(CR₈R₉)_(f)(C═O)(CR₈R₉)_(e)(C₆-C₁₀)aryl,—(CR₈R₉)_(f)(C═O)(CR₈R₉)_(e)(4-10)-membered heterocyclyl,—(CR₈R₉)_(e)O(CR₈R₉)_(f)(C₆-C₁₀)aryl,—(CR₈R₉)_(e)O(CR₈R₉)_(f)(4-10)-membered heterocyclyl,—(CR₈R₉)_(f)S(O)_(d)(CR₈R₉)_(e)(C₆-C₁₀)aryl, and—(CR₈R₉)_(f)S(O)_(d)(CR₈R₉)_(e)(4-10)-membered heterocyclyl;

each of R₃ and R₅ is independently selected from H, cyano, —CF₃, —CHF₂,—CH₂F, (C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, —(C═O)—R₈,—(C═O)—O—R₈, —(C═O)—NR₈R₉, —S(O)_(c)NR₈R₉, —S(O)_(d)(C₁-C₈)alkyl,—(CR₈R₉)_(d)(3-10)-membered cycloalkyl, —(CR₈R₉)_(e)(C₆-C₁₀)aryl,—(CR₈R₉)_(e)(4-10)-membered heterocyclyl,—(CR₈R₉)_(f)(C═O)(CR₈R₉)_(e)(C₆-C₁₀)aryl,—(CR₈R₉)_(f)(C═O)(CR₈R₉)_(e)(4-10)-membered heterocyclyl,—(CR₈R₉)_(e)O(CR₈R₉)_(f)(C₆-C₁₀)aryl,—(CR₈R₉)_(e)O(CR₈R₉)_(f)(4-10)-membered heterocyclyl,—(CR₈R₉)_(f)S(O)_(d)(CR₈R₉)_(e)(C₆-C₁₀)aryl, and—(CR₈R₉)_(f)S(O)_(d)(CR₈R₉)_(e)(4-10)-membered heterocyclyl;

each of the R₃ groups are optionally linked to each other as a 4-12membered mono- or bicyclic ring;

each of the R₅ groups are optionally linked to each other as a 4-12membered mono- or bicyclic ring;

R₄ is H, —CF₃, —CHF₂, —CH₂F, (C₁-C₆)alkyl, (C₂-C₆)alkenyl,(C₂-C₆)alkynyl, —(C═O)—R₈, —(C═O)—O—R₈, —(C═O)—NR₈R₉,—(CR₈R₉)_(d)(3-10)-membered cycloalkyl, —(CR₈R₉)_(e)(C₆-C₁₀)aryl,—(CR₈R₉)_(e)(4-10)-membered heterocyclyl,—(CR₈R₉)_(f)(C═O)(CR₈R₉)_(e)(C₆-C₁₀)aryl,—(CR₈R₉)_(f)(C═O)(CR₈R₉)_(e)(4-10)-membered heterocyclyl,—(CR₈R₉)_(e)O(CR₈R₉)_(f)(C₆-C₁₀)aryl,—(CR₈R₉)_(e)O(CR₈R₉)_(f)(4-10)-membered heterocyclyl,—CR₈R₉)_(f)S(O)_(d)(CR₈R₉)_(e)(C₆-C₁₀)aryl, and—(CR₈R₉)_(f)S(O)_(d)(CR₈R₉)_(e)(4-10)-membered heterocyclyl;

wherein R₆ and R₇ are linked to each other as a 4-12 membered mono- orbicyclic ring;

each of R₈, R₉ and R₁₀ are independently selected from H, (C₁-C₆)alkyl,—(CR₁₁R₁₂)_(e)(3-10)-membered cycloalkyl, —(CR₁₁R₁₂)_(g)(C₆-C₁₀)aryl,and —(CR₁₁R₁₂)_(g)(4-10)-membered heterocyclyl;

any carbon atoms of the (C₁-C₆)alkyl, the (3-10)-membered cycloalkyl,the (C₆-C₁₀)aryl and the (4-10)-membered heterocyclyl of the foregoingR₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, andR₁₆ are independently optionally substituted with 1 to 3 R₁₄substituents each independently selected from halo, cyano, nitro, —CF₃,—CHF₂, —CH₂F, trifluoromethoxy, azido, hydroxyl, —O—R₁₅, (C₁-C₆)alkoxy,(C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, —(C═O)—R₁₁, —(C═O)—R₁₅,—(C═O)—O—R₁₁, —(C═O)—O—R₁₅, —O—(C═O)—R₁₁, —O—(C═O)—R₁₅, —NR11(C═O)—R₁₃,—(C═O)—NR₁₁R₁₂, —(C═O)—NR₁₁R₁₅, —NR₁₁R₁₂, —NR₁₁R₁₅, —NR₁₁OR₁₂,—NR₁₁OR₁₅, —S(O)_(e)NR₁₁R₁₂, —S(O)_(c)NR₁₁R₁₅, —S(O)_(d)(C₁-C₆)alkyl,—S(O)_(d)R₁₅, —O—SO₂—R₁₁, —O—SO₂—R₁₅, —NR₁₁—S(O)_(c), —NR₁₅—S(O)_(c),—(CR₁₁R₁₂)_(e)(3-10)-membered cycloalkyl, —(CR₁₁R₁₂)_(e)(C₆-C₁₀)aryl,—(CR₁₁R₁₂)_(e)(4-10)-membered heterocyclyl,—(CR₁₁R₁₂)_(f)(C═O)(CR₁₁R₁₂)_(e)(C₆-C₁₀)aryl,—(CR₁₁R₁₂)_(f)(C═O)(CR₁₁R₁₂)_(e)(4-10)-membered heterocyclyl,—(CR₁₁R₁₂)_(e)O(CR₁₁R₁₂)_(f)(C₆-C₁₀)aryl,—(CR₁₁R₁₂)_(e)O(CR₁₁R₁₂)_(f)(4-10)-membered heterocyclyl,—(CR₁₁R₁₂)_(f)S(O)_(d)(CR₁₁R₁₂)_(e)(C₆-C₁₀)aryl, and—(CR₁₁R₁₂)_(f)S(O)_(d)(CR₁₁R₁₂)_(e)(4-10)-membered heterocyclyl;

any carbon atoms of the (C₁-C₆)alkyl, the (3-10)-membered cycloalkyl,the (C₆-C₁₀)aryl and the (4-10)-membered heterocyclyl of the foregoingR₁₄ are independently optionally substituted with 1 to 3 R₁₆substituents each independently selected from halo, cyano, nitro, —CF₃,—CHF₂, —CH₂F, trifluoromethoxy, azido, (CH₂)_(e)OH, (C₁-C₆)alkoxy,(C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, —(C═O)—R₁₁, —(C═O)—R₁₅,—(C═O)—O—R₁₁, —(C═O)—O—R₁₅, —O—(C═O)—R₁₁, —O—(C═O)—R₁₅, —NR₁₁(C═O)—R₁₃,—(C═O)—NR₁₁R₁₂, —NR₁₁R₁₂, and —NR₁₁R₁₅;

any nitrogen atoms of the (4-10)-membered heterocyclyl of the foregoingR₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₄, and R₁₅ are independentlyoptionally substituted with (C₁-C₆)alkyl, (C₂-C₆)alkenyl,(C₂-C₆)alkynyl, —(C═O)—R₁₁, —(C═O)—O—R₁₁, —(C═O)—NR₁₁R₁₂,—(CR₁₁R₁₂)_(e)(3-10)- membered cycloalkyl, —(CR₁₁R₁₂)_(e)(C₆-C₁₀)aryl,—(CR₁₁R₁₂)_(e)(4-10)-membered heterocyclyl,—(CR₁₁R₁₂)_(f)(C═O)(CR₁₁R₁₂)_(e)(C₆-C₁₀)aryl, or—(CR₁₁R₁₂)_(f)(C═O)(CR₁₁R₁₂)_(e)(4-10)-membered heterocyclyl;

each R₁₁, R₁₂, and R₁₃ are independently H or (C₁-C₆)alkyl;

R₁₅ is —(CR₁₁R₁₂)_(e)(3-10)-membered cycloalkyl,—(CR₁₁R₁₂)_(e)(C₆-C₁₀)aryl, or —(CR₁₁R₁₂)_(e)(4-10)-memberedheterocyclyl;

a and b are each independently 1, 2, 3, or 4;

c is 1 or 2;

d is 0, 1, or 2; and

e, f, and g are each independently 0, 1, 2, 3, 4, or 5.

In exemplary embodiments of the compounds of formula I, each of A, D, E,G, J, L, M, and Q are C; each of R₁ and R₂ is independently selectedfrom H or halo; R₄ is H or (C₁-C₆)alkyl, R₃ and R₅ are H; R₆ and R₇ arelinked to each other as a 4-12 membered mono- or bicyclic amide ring;R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, a, b, c, d, e, and f are asdefined herein.

In some embodiments, each of A, D, E, G, J, L, M, and Q are C; each ofR₁ and R₂ is independently selected from H or halo; R₄ is H or(C₁-C₆)alkyl, R₃ and R₅ are H; R₆ and R₇ are linked to each other as a4-12 membered mono- or bicyclic urea ring; R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃,R₁₄, R₁₅, R₁₆, a, b, c, d, e, and f are as defined herein.

In some embodiments of the subject matter disclosed herein, the compoundof formula I is the single enantiomer bearing an (R)-configuration atC-3, wherein each of A, D, E, G, J, L, M, and Q are C; each of R₁ and R₂is independently selected from H or halo; R₄ is H or (C₁-C₆)alkyl, R₃and R₅ are H; R₆ and R₇ are linked to each other as a 4-12 memberedmono- or bicyclic amide ring; R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, R₁₆,a, b, c, d, e, and f are as defined herein.

In other embodiments, the compound of formula I is a single enantiomerbearing an (S)-configuration at C-3, wherein each of A, D, E, G, J, L,M, and Q are C; each of R₁ and R₂ is independently selected from H orhalo; R₄ is H or (C₁-C₆)alkyl, R₃ and R₅ are H; R₆ and R₇ are linked toeach other as a 4-12 membered mono- or bicyclic urea ring; R₈, R₉, R₁₀,R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, a, b, c, d, e, and f are as definedherein.

In certain embodiments, the compound may be selected from the groupconsisting of:

-   1-(3-(3,6-difluoro-9H-carbazol-9-yl)-2-hydroxypropyl)-3-fluoropyrrolidin-2-one;-   2-(3-(3,6-difluoro-9H-carbazol-9-yl)-2-hydroxypropyl)-2-azabicyclo[2.2.1]heptan-3-one;-   1-(3-(3,6-difluoro-9H-carbazol-9-yl)-2-hydroxypropyl)imidazolidin-2-one;-   (1R,4S)-2-((R)-3-(3,6-difluoro-9H-carbazol-9-yl)-2-hydroxypropyl)-2-azabicyclo[2.2.1]heptan-3-one;-   (R)-1-(3-(3,6-difluoro-9H-carbazol-9-yl)-2-hydroxypropyl)imidazolidin-2-one;-   (R)-1-((R)-3-(3,6-difluoro-9H-carbazol-9-yl)-2-hydroxypropyl)-3-fluoropyrrolidin-2-one;-   (S)-1-((S)-3-(9H-carbazol-9-yl)-2-hydroxy-2-methylpropyl)-3-fluoropyrrolidin-2-one;-   (R)-1-((R)-3-(9H-carbazol-9-yl)-2-hydroxypropyl)-4-methylimidazolidin-2-one;    or a pharmaceutically acceptable salt or hydrate thereof

The term “pharmaceutically acceptable” as used herein, refers to amaterial, such as a carrier or diluent, which does not abrogate thebiological activity or properties of the compounds described herein, andis relatively nontoxic, i.e., the material may be administered to anindividual without causing undesirable biological effects or interactingin a deleterious manner with any of the components of the composition inwhich it is contained.

The term “pharmaceutically acceptable salt” as used herein, refers tosalts that retain the biological effectiveness of the free acids andbases of the specified compound and that are not biologically orotherwise undesirable. Pharmaceutically acceptable salts of thecompounds of formula I include the acid addition and base salts thereof.Suitable acid addition salts are formed from acids which form non-toxicsalts. Examples include acetate, adipate, arabogalactanesulfonate,ascorbate, aspartate, benzoate, besylate, bicarbonate/carbonate,bisulfate/sulfate, borate, camsylate, cholate, citrate, edisylate,estolate, esylate, formate, fumarate, galacturonate, gluceptate,gluconate, glucuronate, glutamate, hexafluorophosphate, hibenzate,hippurate, hydrochloride/chloride, hydrobromide/bromide,hydroiodide/iodide, 3-hydroxy-2-naphthoate, 1-hydroxy-2-naphthoate,isethionate, lactate, lactobionate, malate, maleate, malonate,mandelate, mesylate, methylsulfate, mucate, napadisylate, naphthalate,2-napsylate, nicotinate, nitrate, oleate, orotate, oxalate, plamitate,pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, saccharate,salicylate, stearate, succinate, sulfosalicylate, tartrate, tosylate,trifluoroacetate, and tryptophanate salts.

Suitable base salts are formed from bases which form non-toxic salts.Examples include adenine, aluminum, 2-amino-2-methylpropan-1-ol,arginine, benethamine, benzathine, calcium, choline, cytosine,diethylamine, diolamine, epolamine, erbumine, ethylenediamine,glucosamine, glycine, guanidine, guanine, hydrabamine, lysine,magnesium, meglumine, morpholine, nicotinamide, olamine, omithine,piperazine, potassium, procaine, proline, pyridoxine, serine, silver,sodium, trolamine, tromethamine, tyrosine, valine and zinc salts. For areview on suitable salts, see “Handbook of Pharmaceutical Salts:Properties, Selection, and Use” by Stahl and Wermuth (Wiley-VCH,Weinheim, Germany, 2002).

A pharmaceutically acceptable salt of a compound of formula I may bereadily prepared by mixing together solutions of the compound of formulaI and the desired acid or base, as appropriate. The salt may precipitatefrom solution and be collected by filtration or may be recovered byevaporation of the solvent. The degree of ionization in the salt mayvary from completely ionized to almost non-ionized.

The compounds of Formula I may also exist in various crystalline forms,known as polymorphs. Polymorphs include the different crystal packingarrangements of the same elemental composition of a compound. Polymorphsmay have different X-ray diffraction patterns, infrared spectra, meltingpoints, density, hardness, crystal shape, optical and electricalproperties, stability, solvates and solubility. Various factors such asthe recrystallization solvent, rate of crystallization, and storagetemperature may cause a single crystal form to dominate.

A “solvate” is intended to mean a pharmaceutically acceptable solvateform of a specified compound that retains the biological effectivenessof such compound. Examples of solvates include compounds of theinvention in combination with water, isopropanol, ethanol, methanol,dimethylsulfoxide, ethyl acetate, acetic acid, or ethanolamine. The term“hydrate” refers to a solvate where the solvent is water. The term“alcoholate” refers to a solvate where the solvent is an alcohol.Hydrates are formed by the combination of one or more molecules of waterwith one molecule of the substance in which the water retains itsmolecular state as H₂O. Non-limiting examples of hydrates includemonohydrates, dihydrates, etc.

The compounds of the invention include compounds of formula I as definedherein, polymorphs, prodrugs, and isomers, thereof (including optical,geometric, and tautomeric isomers) as well as isotopically-labeledcompounds of formula I.

The compounds of the present invention may be administered as prodrugs.Thus certain derivatives of compounds of formula I which may have littleor no pharmacological activity themselves can, when administered into oronto the body, be converted into compounds of formula I having thedesired activity, for example, by hydrolytic cleavage. Such derivativesare referred to as ‘prodrugs’. Further information on the use ofprodrugs may be found in “Pro-drugs as Novel Delivery Systems, Vol. 14,ACS Symposium Series (T. Higuchi and W. Stella) and “BioreversibleCarriers in Drug Design”, Pergamon Press, 1987 (Ed. E. B. Roche,American Pharmaceutical Association). Prodrugs can, for example, beproduced by replacing appropriate functionalities present in thecompounds of formula I with certain moieties known to those skilled inthe art is ‘pro-moieties’ as described, for example, in “Design ofProdrugs” by H. Bundgaard (Elsevier, 1985).

Some examples of such prodrugs include where the compound of formula Icontains a carboxylic acid functionality (—CO₂H), an ester thereof, forexample, replacement of the hydrogen with (C₁-C₈)alkyl; where thecompound of formula I contains an alcohol functionality (—OH), an etherthereof, for example, replacement of the hydrogen with(C₁-C₈)alkanoyloxymethyl; and where the compound of formula I contains asecondary amino functionality (—NHR where R is not H), an amide thereof,for example, replacement of one hydrogen with (C₁-C₁₀)alkanoyl. Furtherexamples of replacement groups in accordance with the foregoing examplesand examples of other prodrug types are known to those of ordinary skillin the art.

Compounds of formula I contain one or more asymmetric carbon atoms. Itis to be understood that all the enantiomers and/or diastereomerscorresponding to the compounds of formula I can be prepared by analogousmethods. All optical isomers and stereoisomers of the compounds offormula I, and mixtures thereof, are considered to be within the scopeof the invention. With respect to the compounds of formula I, theinvention includes the use of a racemate, one or more enantiomericforms, one or more diastereomeric forms, or mixtures thereof. Thecompounds of formula I may also exist as tautomers. This inventionrelates to the use of all such tautomers and mixtures thereof.

Certain functional groups contained within the compounds of the presentinvention can be substituted for bioisosteric groups, that is, groupswhich have similar spatial or electronic requirements to the parentgroup, but exhibit differing or improved physicochemical or otherproperties. Suitable examples are well known to those of skill in theart, and include, but are not limited to moieties described in Patini,et al. Chem Rev. 1996, 96, 3147-3176 and references cited therein.

Included within the scope of the claimed compounds of formula I arepharmaceutically acceptable acid addition or base salts wherein thecounterion is optically active, for example, D-lactate or L-lysine, orracemic, for example, DL-tartrate or DL-arginine. Cis/trans isomers maybe separated by conventional techniques well known to those skilled inthe art, for example, chromatography and fractional crystallization.Conventional techniques for the preparation/isolation of individualenantiomers include chiral synthesis from a suitable optically pureprecursor or resolution of the racemate (or the racemate of a salt orderivative) using, for example, chiral high pressure liquidchromatography (HPLC).

Alternatively, the racemate (or a racemic precursor) may be reacted witha suitable optically active compound, for example, an alcohol, or, inthe case where the compound of formula I contains an acidic or basicmoiety, an acid or base such as tartaric acid or 1-phenylethylamine. Theresulting diastereomeric mixture may be separated by chromatographyand/or fractional crystallization and the diastereomers converted to thecorresponding pure enantiomers and/or diastereomers by means well knownto a skilled person. The chiral compounds of the invention (and chiralprecursors thereof) may be obtained in enantiomerically- and/ordiastereomerically-enriched form using chromatography, typically HPLC,on an asymmetric resin with a mobile phase consisting of a hydrocarbon,typically heptane or hexane, containing from 0 to 50% isopropanol,typically 2 to 20%, and from 0 to 5% of an alkylamine, typically 0.1%diethylamine. Concentration of the eluate affords the enriched mixture.Mixtures of enantiomers and/or diastereomers may be separated byconventional techniques known to those skilled in the art. See, forexample, “Stereochemistry of Organic Compounds” by E. L. Eliel (Wiley,New York, 1994).

The compounds of formula I may be isotopically-labeled, wherein one ormore atoms are replaced by atoms having the same atomic number, but anatomic mass or mass number different from the atomic mass or mass numberusually found in nature. Examples of isotopes suitable for inclusion inthe compounds of the invention include isotopes of hydrogen, such as ²Hand ³H, carbon, such as ¹¹C, ¹³C and ¹⁴C, chlorine, such as ³⁶Cl,fluorine, such as ¹⁸F, iodine, such as ¹²³I and ¹²⁵I, nitrogen, such as¹³N and ¹⁵N, oxygen, such as ¹⁵O, ¹⁷O and ¹⁸O, phosphorous, such as ³²P,and sulfur, such as ³⁵S. Certain isotopically-labeled compounds offormula I, for example, those incorporating a radioactive isotope, areuseful in drug and/or substrate tissue distribution studies. Theradioactive isotopes tritium, i.e. ³H, and carbon-14, i.e. ¹⁴C, areparticularly useful for this purpose in view of their ease ofincorporation and ready means of detection. Substitution with heavierisotopes such as deuterium, i.e. ²H, may afford certain therapeuticadvantages resulting from greater metabolic stability, for example,increased in vivo half-life or reduced dosage requirements, and hencemay be preferred in some circumstances. Substitution with positronemitting isotopes, such as ¹¹C, ¹⁸F, ¹⁵O and ¹³N, can be useful inPositron Emission Topography (PET) studies for examining substratereceptor occupancy. Isotopically-labeled compounds of formula I can begenerally prepared by conventional techniques known to those skilled inthe art or by processes analogous to those described in the accompanyingExamples and Preparations using appropriate isotopically-labeledreagents in place of the non-labeled reagent previously employed.

The compounds of the present invention modulate Cry1 and/or Cry2. Asused herein, “modulating” refers to increasing, decreasing, or alteringCry1 and Cry2 function, activity or intrinsic characteristics.Modulation of Cry1 or Cry2 includes any one of the following: binding toCry1 or Cry2; inhibiting modification of Cry1 or Cry2; altering Cry1 orCry2 localization; increasing or decreasing Cry1 or Cry2 stabilization;increasing or decreasing the binding between Cry1 or Cry2 to a target;increasing or decreasing Cry1 or Cry2 activity; and increasing ordecreasing activity of a Cry1 or Cry2 target.

Modulation of Cry1 and Cry2 includes binding of a compound of thepresent invention to Cry1 and/or Cry2, either through direct interactionor indirect interaction. In some aspects, a compound of the presentinvention may bind to a complex containing Cry1 and/or Cry2. Methods fordetecting interaction between small molecules and proteins are known inthe art, for example, immunoprecipitation techniques, chromatography,and various array formats.

Intrinsic characteristics of Cry1 and Cry2, such as post-translationalmodification, stability, or localization, may be altered by thecompounds of the present invention. Post-translational modification ofCry1 and Cry2 may play a critical role in determining the activity,stability, or cellular localization of Cry1 and Cry2. Some studies haveshown that phosphorylation may alter Cry1 and Cry2 stability. Compoundsof the present invention may prevent or increase post-translationalmodification of Cry1 and Cry2, for example, phosphorylation,ubiquitination, acetylation, glycosylation, ribosylation, orsumoylation.

Methods for detecting post-translational modification of Cry1 or Cry2can be readily performed by one skilled in the art. Such methods ofdetection include western blot and radioimmunoassays. Cry1 and Cry2localize to the nucleus under particular conditions, for example, uponheterodimerization with Per1 and Per2. Once within the nucleus, Cry1 andCry2 play a role in disrupting the nuclear CLOCK-BMAL1 complex frominitiating transcription, thereby downregulating circadian rhythm genesin a negative feedback loop that is crucial for maintaining circadianoscillations. Localization of proteins can be readily determined by onein the art, for example, by immunofluorescence, subcellularfractionation and western blot assays. Downregulation of Cry1 and Cry2is also critical for circadian oscillations, and is mediated at thetranscriptional and protein level. Cry1 and Cry2 stability can bemeasured by methods known in the art, as well as those presented inExamples 5-8.

Cry1 and Cry2 activity, as used herein, includes the binding betweenCry1 or Cry2 to a target and the activity of a downstream Cry1 or Cry2target. Compounds of the present invention may increase or decrease thebinding between Cry1 or Cry2 to a target. Targets that bind to Cry1and/or Cry2 are known in the art, and include Per1, Per2, glucocorticoidreceptor, the CLOCK-BMAL1 promoter sequence, and the VEGF promotersequence. Other targets include genes for which expression is modulatedby Cry1 or Cry2, including genes that contain an E-box sequence in theirpromoter. Such genes include, without limitation, Dbp, Rev-erb alpha,Rev-erb beta, Ror alpha, Ror beta, Ror gamma, Per1, Per2, Per3, Cry1,Cry2, Pck1, G6Pc, Avp, Vip, Cck, SP (substance P), AA-Nat, PK2(Prokinectin 2), c-Myc, MyoD and Nampt. Cry1 and Cry2 targets referencedherein also include those targets that have yet to be identified.

Binding between Cry1 or Cry2 and targets can be determined by, forexample, immunoprecipitation, yeast two-hybrid, affinity chromatography.Downstream activity of Cry1 or Cry2 targets comprisesCLOCK-BMAL1-mediated transcription, binding of Cry1 or Cry2 to theCLOCK-BMAL-1 promoter, binding of Cry1 or Cry2 to the VEGF promoter,Per1 or Per2 localization or stability, CLOCK-BMAL1 dimerization,expression of CLOCK-BMAL1 target genes, such as Cry1, Cry2, Per1, Per2,Rev-erb α and β, Rora, TIM proteins, and VEGF. Methods for detectingpromoter activity can be determined by chromatin immunoprecipitation,electrophoretic mobility shift assay, or promoter-luciferase assays asdescribed in Examples 3 and 4. Methods for determining expression oftarget genes include gene expression analysis and microarrays, which canbe readily performed by one ordinarily skilled in the art.

In some embodiments, methods or assays for determining putative efficacymay be useful to identify particular compounds of those described hereinthat are suitable for treating or alleviating a symptom of aCry-mediated disease or disorder. In one aspect, the concentration ofthe compound to induce a response halfway between the baseline andmaximum after a specified exposure time (referred to herein as the EC₅₀value or concentration) can be determined in an in vitro assay thatmeasures the effect of the compound on core clock gene expression.Luciferase reporters operably linked to core clock gene promotersequences (i.e., Per1, Per2, Cry1, Cry2 or Bmal1) are introduced intocells (i.e., transfection, transduction, infection) that are treatedwith the compounds of the present invention, and the luminescence (orclock gene-driven expression) is measured over time. Specifically, theperiod, amplitude, and phase of the luminescence compared to expectedexpression correlating to the circadian rhythm is determined. The EC₅₀value for a compound can be calculated using methods readily availableto the skilled person in the art. An example of such an assay isdescribed herein in Example 3. The EC₅₀ values are useful for assessingthe potency of the compounds of the present invention.

In other aspect, the compounds of the present invention with increasedefficacy can be determined by in vivo assay. The compounds areadministered to a subject (i.e., a mouse model) for a period of time. Abiological sample is isolated from the subject, and the concentrationlevels of the compound present in the biological sample is measured. Thebiological sample is, for example, whole blood or any fraction thereof(i.e., serum or plasma), or a tissue, such as a tissue that is affectedby a Cry-mediated disease or disorder. The concentration detected in thesample of the treated subject is compared to the EC₅₀ concentrationvalue for the same compound, as tested in a relevant in vitro assay (asdescribed above and in Example 3). Compounds with a measured compoundconcentration in vivo greater than the determined EC50 value arepreferred compounds of the present invention. These preferred compoundsmay demonstrate increased efficacy for treating or alleviating a symptomof a Cry-mediated disease or disorder.

In other aspects of the subject matter disclosed herein, apharmaceutical composition is provided, comprising the compoundaccording to formula I and a pharmaceutically acceptable carrier,adjuvant or diluent. Methods of preparing various pharmaceuticalcompositions with a specific amount of active compound are known, orwill be apparent, to those skilled in the art. In addition, those ofordinary skill in the art are familiar with formulation andadministration techniques. Such topics will be discussed, e.g. inGoodman and Gilman's “The Pharmaceutical Basis of Therapeutics”, currentedition, Pergamon Press; and “Remington's Pharmaceutical Sciences”,current edition, Mack Publishing, Co., Easton, Pa. These techniques canbe employed in appropriate aspects and embodiments of the methods andcompositions described herein. Pharmaceutical compositions arepreferably manufactured under GMP conditions. The following examples areprovided for illustrative purposes only and are not meant to serve aslimitations of the present invention.

Because the compounds described herein are intended for use inpharmaceutical compositions, it will readily be understood that they areeach preferably provided in substantially pure form, for example atleast 50% pure, at least 55% pure, at least 60% pure, at least 65% pure,at least 70% pure, at least 75% pure, at least 80% pure, at least 85%,at least 90% pure, at least 95% pure, at least 96% pure, at least 97%pure, at least 98% pure, or at least 99% pure. Percentages as providedherein are on a weight for weight basis. Impure preparations of thecompounds may be used for preparing the more pure forms used in thepharmaceutical compositions; these less pure preparations of thecompounds should contain at least 1%, more suitably at least 5%, e.g. 10to 49% of a compound of the Formula I.

The compounds of Formula I may be provided in suitable topical, oral,nasal, ocular, mucosal, rectal, vaginal, and parenteral pharmaceuticalformulations for use in the treatment of Cry mediated diseases. Thecompounds of the present invention may be administered orally as tabletsor capsules, as oily or aqueous suspensions, lozenges, troches, powders,granules, emulsions, syrups or elixirs. The compositions for oral usemay include one or more agents for flavoring, sweetening, coloring andpreserving in order to produce pharmaceutically elegant and palatablepreparations. Tablets may contain pharmaceutically acceptableexcipients, carriers, diluents, and adjuvants as an aid in themanufacture of such tablets. As is conventional in the art, thesetablets may be coated with a pharmaceutically acceptable entericcoating, such as glyceryl monostearate or glyceryl distearate, to delaydisintegration and absorption in the gastrointestinal tract to provide asustained action over a longer period. The dissolution rate of poorlywater-soluble compounds may be enhances by the use of spray-drieddispersion, such as those described by Takeuchi, H. et al. J. Pharm.Pharmacol. 1987, 39, 769-773.

Formulations for oral use may be in the form of hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin. They may alsobe in the form of soft gelatin capsules wherein the active ingredient ismixed with water or an oil medium, such as peanut oil, liquid paraffinor olive oil.

Aqueous suspensions normally contain active ingredients in admixturewith excipients suitable for the manufacture of an aqueous suspension.Such excipients may be a suspending agent, such as Kolliphor, sodiumcarboxymethyl cellulose, methyl cellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragancanth andgum acacia; a dispersing or wetting agent that may be a naturallyoccurring phosphatide such as lecithin, a condensation product ofethylene oxide and a long chain fatty acid, for example polyoxyethylenestearate, a condensation product of ethylene oxide and a long chainaliphatic alcohol such as heptadecaethyleneoxycetanol, a condensationproduct of ethylene oxide and a partial ester derived from a fatty acidand hexitol such as polyoxyethylene sorbitol monooleate or a fatty acidhexitol anhydrides such as polyoxyethylene sorbitan monooleate.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleaginous suspension. This suspension may beformulated according to known methods as aqueous isotonic solutions orsuspensions, and suppositories can be prepared from fatty emulsions orsuspensions. The compositions can be sterilized and/or containadjuvants, such as preserving, stabilizing, wetting or emulsifyingagents, solution promoters, salts for regulating the osmotic pressureand/or buffers. In addition, they can also contain other therapeuticallyvaluable substances. The sterile injectable preparation may also beformulated as a suspension in a non-toxin parenterally-acceptablediluent or solvent, for example as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringers solution and isotonic sodium chloride solution. For this purposeany bland fixed oil may be employed including synthetic mono- ordiglycerides. In addition fatty acids such as oleic acid find use in thepreparation of injectables.

The compounds of formula I may also be administered in the form ofsuppositories for rectal administration of the drug. These compositionscan be prepared by mixing the drug with a suitable non-irritatingexcipient that is solid at about 25° C., but liquid at rectaltemperature and will therefore melt in the rectum to release the drug.Such materials include cocoa butter and other glycerides.

For topical or transdermal use preparations, for example, creams,ointments, jellies solutions or suspensions containing the compounds ofthe present invention are employed. Suitable formulations fortransdermal applications include an effective amount of a compound ofthe present invention with a carrier. A carrier can include absorbablepharmacologically acceptable solvents to assist passage through the skinof the host. For example, transdermal devices are in the form of abandage comprising a backing member, a reservoir containing the compoundoptionally with carriers, optionally a rate controlling barrier todeliver the compound to the skin of the host at a controlled andpredetermined rate over a prolonged period of time, and means to securethe device to the skin. Matrix transdermal formulations andiontophoresis devices can also be used. Suitable formulations fortopical application, e.g., to the skin and eyes, are preferably aqueoussolutions, ointments, creams or gels well-known in the art. Such cancontain solubilizers, stabilizers, tonicity enhancing agents, buffersand preservatives.

The active compounds can be prepared with pharmaceutically acceptablecarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art.

The compounds of formula I may also be prepared in the form of liposomedelivery systems such as small unilamellar vesicles, large unilamellarvesicles and multimellar vesicles. Liposomes can be formed from avariety of phospholipids, such as cholesterol, stearylamine orphosphatidylcholines.

Suitable extended release form of the either active pharmaceuticalingredient or both may be a matrix tablet or capsule composition.Suitable matrix forming materials include, for example, waxes (e.g.,carnauba, bees wax, paraffin wax, ceresine, shellac wax, fatty acids,and fatty alcohols), oils, hardened oils or fats (e.g., hardenedrapeseed oil, castor oil, beef tallow, palm oil, and soya bean oil), andpolymers (e.g., hydroxypropyl cellulose, polyvinylpyrrolidone,hydroxypropyl methyl cellulose, and polyethylene glycol). Other suitablematrix tableting materials are microcrystalline cellulose, powderedcellulose, hydroxypropyl cellulose, ethyl cellulose, with othercarriers, and fillers. Tablets may also contain granulates, coatedpowders, or pellets. Tablets may also be multi-layered. Multi-layeredtablets are especially preferred when the active ingredients havemarkedly different pharmacokinetic profiles. Optionally, the finishedtablet may be coated or uncoated.

The coating composition typically contains an insoluble matrix polymer(approximately 15-85% by weight of the coating composition) and a watersoluble material (e.g., approximately 15-85% by weight of the coatingcomposition). Optionally an enteric polymer (approximately 1 to 99% byweight of the coating composition) may be used or included. Suitablewater soluble materials include polymers such as polyethylene glycol,hydroxypropyl cellulose, hydroxypropyl methyl cellulose,polyvinylpyrrolidone, polyvinyl alcohol, and monomeric materials such assugars (e.g., lactose, sucrose, fructose, mannitol and the like), salts(e.g., sodium chloride, potassium chloride and the like), organic acids(e.g., fumaric acid, succinic acid, lactic acid, and tartaric acid), andmixtures thereof. Suitable enteric polymers include hydroxypropyl methylcellulose, acetate succinate, hydroxypropyl methyl cellulose, phthalate,polyvinyl acetate phthalate, cellulose acetate phthalate, celluloseacetate trimellitate, shellac, zein, and polymethacrylates containingcarboxyl groups.

The coating composition may be plasticized according to the propertiesof the coating blend such as the glass transition temperature of themain component or mixture of components or the solvent used for applyingthe coating compositions. Suitable plasticizers may be added from 0 to50% by weight of the coating composition and include, for example,diethyl phthalate, citrate esters, polyethylene glycol, glycerol,acetylated glycerides, acetylated citrate esters, dibutylsebacate, andcastor oil. If desired, the coating composition may include a filler.The amount of the filler may be 1% to approximately 99% by weight basedon the total weight of the coating composition and may be an insolublematerial such as silicon dioxide, titanium dioxide, talc, kaolin,alumina, starch, powdered cellulose, MCC, or polacrilin potassium. Thecoating composition may be applied as a solution or latex in organicsolvents or aqueous solvents or mixtures thereof. If solutions areapplied, the solvent may be present in amounts from approximate by25-99% by weight based on the total weight of dissolved solids. Suitablesolvents are water, lower alcohol, lower chlorinated hydrocarbons,ketones, or mixtures thereof. If latexes are applied, the solvent ispresent in amounts from approximately 25-97% by weight based on thequantity of polymeric material in the latex. The solvent may bepredominantly water.

Dosage levels of the compounds of the present invention are of the orderof about 0.5 mg/kg body weight to about 100 mg/kg body weight, or anyincrement in between. A preferred dosage rate is between about 30 mg/kgbody weight to about 100 mg/kg body weight. The total daily dose may beadministered in single or divided doses. Suitable therapeutic doses ofthe compounds of formula I may be in the range of 1 microgram (m) to1000 milligrams (mg) per kilogram body weight of the recipient per day,and any increment in between, such as, e.g., 1, 2, 3, 5, 10, 25, 50, 75,100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 μg (1 mg); 2, 3, 5,10, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 mg.It will be understood, however, that specific dose level for anyparticular patient will depend upon a number of factors including theactivity of the particular compound being administered, the age, bodyweight, general health, sex, diet, time of administration, route ofadministration, rate of excretion, drug combination and the severity ofthe particular disease undergoing therapy.

Dosage regimens may be adjusted to provide the optimum desired response.For example, a single bolus may be administered, several divided dosesmay be administered over time or the dose may be proportionally reducedor increased as indicated by the exigencies of the therapeuticsituation. It is especially advantageous to formulate parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form, as used herein, refers tophysically discrete units suited as unitary dosages for mammaliansubjects to be treated; each unit containing a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the therapeutic agent andthe particular therapeutic or prophylactic effect to be achieved, and(b) the limitations inherent in the art of compounding such an activecompound for the treatment of sensitivity in individuals. Thus, theskilled artisan would appreciate, based upon the disclosure providedherein, that the dose and dosing regimen is adjusted in accordance withmethods well-known in the therapeutic arts. That is, the maximumtolerable dose can be readily established, and the effective amountproviding a detectable therapeutic benefit to a patient may also bedetermined, as can be temporal requirements for administering each agentto provide a detectable therapeutic benefit to the patient. Accordingly,while certain dose and administration regimens are exemplified herein,these examples in no way limit the dose and administration regimen thatmay be provided to a patient in practicing the present invention.

In some embodiments, the compounds and compositions described herein areadministered to a subject around bed time or sleep time at night.Preferably, the compounds and compositions described herein areadministered to the subject within 6 hours, 5 hours, 4 hours, 3 hours,120 minutes, 90 minutes, 60 minutes, 45 minutes, 30 minutes, 25 minutes,20 minutes, 15 minutes, 10 minutes, 5 minutes, or immediately before orafter bed time. Preferably, the compounds and compositions of thepresent invention are administered to the subject within 2 hours toimmediately before bed time. “Bed time”, as used herein, refers to thetime of night at which a subject goes to bed with the intention ofresting or falling asleep.

In other embodiments, the compounds and compositions described hereinmay be administered with or without food. When the compounds orcompositions are administered with food, it may be preferable toadminister the compounds or compositions within 4 hours before or aftera meal, such as breakfast, lunch, dinner or a snack. For example, thecompounds or compositions of the present invention are administeredwithin 6 hours, within 5 hours, within 4 hours, within 3 hours, within120 minutes, within 90 minutes, within 60 minutes, within 45 minutes,within 30 minutes, within 25 minutes, within 20 minutes, within 15minutes, within 10 minutes, within 5 minutes, or immediately before orafter a meal. Preferably, the compounds and compositions of the presentinvention are administered to the subject within 4 hours, within 3hours, within 120 minutes, within 90 minutes, or within 60 minutes afterdinner.

In another aspect of the subject matter disclosed herein, a method oftreating a Cry-mediated disease or disorder is provided, comprisingadministering a therapeutically effective amount of a compound accordingto formula I as described in any of the preceding embodiments herein. Apreferred embodiment of the present invention is the method of treatinga Cry-mediated disease or disorder wherein the disease or disordercharacterized by abnormal levels of Cry is selected from the groupconsisting of diabetes, complications associated with diabetes,metabolic syndrome, insulin resistance syndrome, obesity, glaucoma,Cushing's syndrome, inflammatory disorders, mitochondrial disorders,Friedrich's ataxia, psychotic depression, Alzheimer's disease,neuropathic pain, drug abuse, osteoporosis, cancer, maculardegeneration, and myopathy. Particularly preferred Cry-mediated diseasesor disorders treated by the compounds disclosed herein include diabetes,diabetic complications such as diabetic neuropathy, diabeticretinopathy, diabetic nephropathy, cataract formation, glaucoma,diabetic angiopathy, atherosclerosis; nonalcoholic steatohepatitis(NASH); non-alcoholic fatty liver disease (NAFLD); asthma; and chronicobstructive pulmonary disease (COPD).

The terms “administer,” “administering”, “administration,” and the like,as used herein, refer to the methods that may be used to enable deliveryof compounds or compositions to the desired site of biological action.These methods include, but are not limited to oral, parenteral, topical,mucosal, ocular, ophthalmic, vaginal, and rectal administration. Thoseof skill in the art are familiar with administration techniques that canbe employed with the compounds and methods described herein, e.g., asdiscussed in Goodman and Gilman, The Pharmacological Basis ofTherapeutics, current ed.; Pergamon; and Remington's, PharmaceuticalSciences (current edition), Mack Publishing Co., Easton, Pa. As usedherein, “parenteral administration” of a pharmaceutical compositionincludes any route of administration characterized by physical breachingof a tissue of a subject and administration of the pharmaceuticalcomposition through the breach of the tissue. Parenteral administrationthus includes, but is not limited to, administration of a pharmaceuticalcomposition by injection of the composition, by application of thecomposition through a surgical incision, by application of thecomposition through a tissue-penetrating non-surgical wound, and thelike. In particular, parenteral administration is contemplated toinclude, but is not limited to, subcutaneous, intraperitoneal,intramuscular, and intrasternal injection, and kidney dialytic infusiontechniques.

A “subject” in the context of the present invention is preferably amammal. The mammal can be a human, non-human primate, mouse, rat, dog,cat, horse, or cow, but are not limited to these examples. Mammals otherthan humans can be advantageously used as subjects that represent animalmodels of a Cry-mediated disease or disorder, such as ob/ob mice. Asubject can be male or female. A subject can be one who has beenpreviously diagnosed or identified as having a Cry-mediated disease ordisorder, and optionally has already undergone, or is undergoing, atherapeutic intervention or treatment for the disease or disorder.Alternatively, a subject can also be one who has not been previouslydiagnosed as having a Cry-mediated disease or disorder. For example, asubject can be one who exhibits one or more risk factors for aCry-mediated disease or disorder, or a subject who does not exhibit riskfactors for a Cry-mediated disease or disorder, or a subject who isasymptomatic for a Cry-mediated disease or disorder. A subject can alsobe one who is suffering from or at risk of developing a Cry-mediateddisease or disorder, or who is suffering from or at risk of developing arecurrence of a Cry-mediated disease or disorder. A subject can also beone who has been previously treated for a Cry-mediated disease ordisorder, whether by administration of the compounds and compositionsdisclosed herein, either alone or in combination with other therapeuticagents, surgery, or any combination of the foregoing. The term “subject”and “patient”, as used herein, may be used interchangeably.

A “Cry-mediated disease or disorder” may include, without limitation,diabetes (including, without limitation, insulin-dependent “Type I”diabetes, non-insulin dependent “Type II” diabetes, gestationaldiabetes, and diabetes-related complications like diabetic neuropathy,diabetic retinopathy, diabetic cardiomyopathy, diabetic nephropathy,periodontal disease, and diabetic ketoacidosis), metabolic syndrome,insulin resistance syndrome, obesity, glaucoma, Cushing's syndrome,psychotic depression, Alzheimer's disease, neuropathic pain, drug abuse,osteoporosis, cancer, macular degeneration, and myopathy.

The term “treating”, “treat”, or “treatment” as used herein includespreventative (e.g. prophylactic), palliative, adjuvant, and curativetreatment. For example, the treatment of type 2 diabetes, as used hereinmeans that a patient having type 2 diabetes or at risk of having type 2diabetes can be treated according to the methods described herein. Forpatients undergoing preventative treatment, a resulting reduction in theincidence of the disease state being preventively treated is themeasurable outcome of the preventative treatment.

The term “alleviating” or “alleviate” as used herein describes a processby which the severity of a sign or symptom of a disorder is decreased,reduced, or inhibited. Importantly, a symptom can be alleviated withoutbeing eliminated. In a preferred embodiment, the administration ofpharmaceutical compositions of the invention leads to the elimination ofa symptom, however, elimination is not required. Therapeuticallyeffective amounts of the compounds or pharmaceutical compositionsdescribed herein are expected to decrease the severity of a symptom.

As used herein the term “symptom” is defined as an indication ofdisease, illness, injury, or that something is not right in the body.Symptoms are felt or noticed by the individual experiencing the symptom,but may not easily be noticed by others. Others are defined byhealth-care or clinical professionals.

The term “metabolic syndrome”, as used herein, unless otherwiseindicated means psoriasis, diabetes mellitus, wound healing,inflammation, neurodegenerative diseases, galactosemia, maple syrupurine disease, phenylketonuria, hypersarcosinemia, thymine uraciluria,sulfinuria, isovaleric academia, saccharopurinuria, 4-hydroxybutyricaciduria, glucose-6-phosphate dehydrogenase deficiency, and pyruvatedehydrogenase deficiency.

The term “obesity” or “obese”, as used herein, refers generally toindividuals who are at least about 20-30% over the average weight forhis/her age, sex and height. Technically, “obese” is defined, for males,as individuals whose body mass index is greater than 27.8 kg/m², and forfemales, as individuals whose body mass index is greater than 27.3kg/m². Those of skill in the art readily recognize that the inventionmethod is not limited to those who fall within the above criteria.Indeed, the method of the invention can also be advantageously practicedby individuals who fall outside of these traditional criteria, forexample, by those who may be prone to obesity.

The term “inflammatory disorders”, as used herein, refers to disorderssuch as asthma, chronic obstructive pulmonary disease (COPD), rheumatoidarthritis, ankylosing spondylitis, psoriatic arthritis, psoriasis,chondrocalcinosis, gout, inflammatory bowel disease, ulcerative colitis,Crohn's disease, fibromyalgia, and cachexia.

The term “Cushing's syndrome”, as used herein, refers to a constellationof signs and symptoms resulting from prolonged exposure to elevatedlevels of cortisol. Cushing's syndrome may result from endogenous orexogenous causes. Causes of endogenous Cushing's syndrome includepituitary tumors (also called Cushing's disease), adrenal tumors, andectopic secretion of adrenocorticotropic hormone (ACTH) and/orcorticotropin-releasing hormone (CRH) from other tumors (including, butnot limited to, small cell lung cancer). Exogenous (or iatrogenic)Cushing's syndrome results from use of corticosteroids for the treatmentof a variety of disorders, including inflammatory disorders including,but not limited to, asthma, psoriasis and rheumatoid arthritis.

The term “mitochondrial diseases”, as used herein, refers to diseasessuch as mitochondrial encephalomyopathy, lactic acidosis, andstroke-like episodes (MELAS), myoclonic epilepsy with ragged-red fibers(MERRF), Kearns-Sayre syndrome, chronic progressive externalophthalmoplegia, Leber's hereditary optic neuropathy, Leigh syndrome,diabetes, deafness, neurogenic muscle weakness, ataxia, and retinitispigmentosa (NARP), and myoneurogenic gastrointestinal encephalopathy.

The term “cancer”, as used herein, refers to disorders and diseasescharacterized by uncontrolled cell growth and/or proliferation, andinclude benign and malignant cancers, hyperproliferative disorders anddiseases, and metastases. Examples of particularly preferred cancersinclude solid tumor cancers or epithelial cancers, including but notlimited to: lung cancer; brain cancer; pancreatic cancer; head and neckcancer (e.g., squamous cell carcinoma); breast cancer; colorectalcancer; liver cancer; stomach cancer; kidney cancer; ovarian cancer;prostate cancer; or an adenocarcinoma. Other cancers are those withincreased VEGF expression, increased angiogenesis, or hypoxic tumors.

The phrase “therapeutically effective amount”, as used herein, refers tothe amount of drug or pharmaceutical agent that will elicit thebiological or medical response of a tissue, system, animal, or humanthat is being sought by a researcher, veterinarian, medical doctor orother.

The phrase “amount . . . effective to lower blood glucose levels”, asused herein, refers to levels of compound sufficient to providecirculating concentrations high enough to accomplish the desired effect.Such a concentration typically falls in the range of about 10 nM up to 2μM; with concentrations in the range of about 100 nM up to 500 nM beingpreferred. As noted previously, since the activity of differentcompounds which fall within the definition of formula I as set forthabove may vary considerably, and since individual subjects may present awide variation in severity of symptoms, it is up to the practitioner todetermine a subject's response to treatment and vary the dosagesaccordingly.

The phrase “insulin resistance”, as used herein, refers to the reducedsensitivity to the actions of insulin in the whole body or individualtissues, such as skeletal muscle tissue, myocardial tissue, fat tissueor liver tissue. Insulin resistance occurs in many individuals with orwithout diabetes mellitus.

The phrase “insulin resistance syndrome”, as used herein, refers to thecluster of manifestations that include insulin resistance,hyperinsulinemia, non-insulin dependent diabetes mellits (NIDDM),arterial hypertension, central (visceral) obesity, and dyslipidemia. Thecompounds of the present invention may also be useful in the treatmentof other metabolic disorders associated with impaired glucoseutilization and insulin resistance including major late-stagecomplications of NIDDM, such as diabetic angiopathy, atherosclerosis,non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease(NAFLD), diabetic nephropathy, diabetic neuropathy, and diabetic ocularcomplications such as retinopathy, cataract formation and glaucoma, andmany other complications linked to NIDDM, including dyslipidemia,glucocorticoid-induced insulin resistance, polycystic ovarian syndrome,obesity, hyperglycemia, hyperlipidemia, hypercholesteremia,hypertriglyceridemia, hyperinsulinemia, and hypertension. Briefdescriptions of these conditions are available in any medicaldictionary, for instance, “Stedman's Medical Dictionary” (Xth Ed.).

Compounds and compositions disclosed herein can be administered intherapeutically effective amounts in combination with one or moreadditional therapeutic agents as defined herein. For example,synergistic effects can occur with other substances used in thetreatment of Cry-mediated diseases or disorders. Where the compounds ofthe invention are administered in conjunction with other therapies,dosages of the co-administered compounds will of course vary dependingon the type of co-drug employed, on the specific drug employed, on thecondition being treated and so forth.

As used herein, the terms “combination treatment”, “combinationtherapy”, “combined treatment” or “combinatorial treatment”, usedinterchangeably, refer to a treatment of an individual with at least twodifferent therapeutic agents. The terms “co-administration” or “combinedadministration” or the like as utilized herein are meant to encompassadministration of the selected therapeutic agents to a single subject,and are intended to include treatment regimens in which the agents arenot necessarily administered by the same route of administration or atthe same time. The combination treatments described herein are intendedto provide the beneficial effect from the co-action of the compounds andcompositions disclosed herein and one or more additional therapeuticagents. The beneficial effect of the combination includes, but is notlimited to, pharmacokinetic or pharmacodynamic co-action resulting fromthe combination of the compounds disclosed herein and the therapeuticagents. Administration of these therapeutic agents in combinationtypically is carried out over a defined time period (usually minutes,hours, days or weeks depending upon the combination selected). In someembodiments, the compounds of the present invention are administered incombination with one or more additional therapeutic agent in asimultaneous or sequential manner. When administered simultaneously, thecompound(s) of the present invention may be administered in, forexample, the same capsule as that of an additional therapeutic agent.Alternatively, the compound of the present invention and the additionaltherapeutic agent are encompassed in separate compositions (i.e.,capsules) that are to be administered at the same time. Whenadministered sequentially, the compound(s) of the present invention maybe administered prior to or after the administration of the additionaltherapeutic agent. The term “pharmaceutical combination” means a productthat results from the mixing or combining of more than one activeingredient and includes both fixed and non-fixed combinations of theactive ingredients. A “fixed combination” means that the activeingredients, e.g. a compound as disclosed herein and an additionaltherapeutic agent, are both administered to a patient simultaneously inthe form of a single entity or dosage. A “non-fixed combination” meansthat the active ingredients, e.g. a compound as disclosed herein and anadditional therapeutic agent, are both administered to a patient asseparate entities either simultaneously, concurrently or sequentiallywith no specific time limits, wherein such administration providestherapeutically effective levels of the 2 compounds in the body of thepatient. The latter also applies to cocktail therapy, e.g. theadministration of 3 or more active ingredients.

Therapeutic agents for treating diabetes, metabolic syndrome, obesity,insulin resistance syndrome, diabetic complications or cancer include,without limitation of the following, insulin, hypoglycemic agents,anti-diabetic agents, anti-inflammatory agents, lipid reducing agents,anti-hypertensives such as calcium channel blockers, β-adrenergicreceptor blockers, cyclooxygenase-2 inhibitors, angiotensin systeminhibitors, ACE inhibitors, renin inhibitors, chemotherapeutic agents,radiotherapy, hormone-modulating agents, immunomodulating agents,anti-angiogenic agents, together with other common risk factor modifyingagents.

Insulin includes rapid acting forms, such as Insulin lispro rDNA origin:HUMALOG (1.5 mL, 10 mL, Eli Lilly and Company, Indianapolis, Ind.),Insulin Injection (Regular Insulin) form beef and pork (regular ILETINI, Eli Lilly], human: rDNA: HUMULIN R (Eli Lilly), NOVOLIN R (NovoNordisk), Semisynthetic: VELOSULIN Human (Novo Nordisk), rDNA Human,Buffered: VELOSULIN BR, pork: regular Insulin (Novo Nordisk), purifiedpork: Pork Regular ILETIN II (Eli Lilly), Regular Purified Pork Insulin(Novo Nordisk), and Regular (Concentrated) ILETIN II U-500 (500units/mL, Eli Lilly); intermediate-acting forms such as Insulin ZincSuspension, beef and pork: LENTE ILETIN G I (Eli Lilly), Human, rDNA:HUMULIN L (Eli Lilly), NOVOLIN L (Novo Nordisk), purified pork: LENTEILETIN II (Eli Lilly), Isophane Insulin Suspension (NPH): beef and pork:NPH ILETIN I (Eli Lilly), Human, rDNA: HUMULIN N (Eli Lilly), Novolin N(Novo Nordisk), purified pork: Pork NPH Iletin II (Eli Lilly), NPH-N(Novo Nordisk); and long-acting forms such as Insulin zinc suspension,extended (ULTRALENTE, Eli Lilly), human, rDNA: HUMULIN U (Eli Lilly).

Hypoglycemic agents include, without limitation, sulfonylureas:Acetohexamide (Dymelor), Chlorpropamide (Diabinese), Tolbutamide(Orinase); second-generation sulfonylureas: Glipizide (Glucotrol,Glucotrol XL), Glyburide (Diabeta; Micronase; Glynase), Glimepiride(Amaryl); Biguanides: Metformin (Glucophage); α-glucosidase inhibitors:Acarbose (Precose), Miglitol (Glyset), Thiazolidinediones: Rosiglitazone(Avandia), Pioglitazone (Actos), Troglitazone (Rezulin); Meglitinides:Repaglinide (Prandin); and other hypoglycemics such as Acarbose;Buformin; Butoxamine Hydrochloride; Camiglibose; Ciglitazone;Englitazone Sodium; Darglitazone Sodium; Etoformin Hydrochloride;Gliamilide; Glibomuride; Glicetanile Gliclazide Sodium; Gliflumide;Glucagon; Glyhexamide; Glymidine Sodium; Glyoctamide; Glyparamide;Linogliride; Linogliride Fumarate; Methyl Palmoxirate; PalmoxirateSodium; Pirogliride Tartrate; Proinsulin Human; Seglitide Acetate;Tolazamide; Tolpyrramide; Zopolrestat.

Anti-diabetic agents include, without limitation, dipeptidyl peptidaseIV inhibitors such as sitagliptin, alogliptin, vildagliptin,saxagliptin, linagilptin, anagliptin, teneligliptin, gemigliptin,dutogliptin, or any other gliptins currently in development, berberineand lupeol; and GLP-1 agonists such as exenatide, liraglutide,albiglutide, taspogenitde, and AVE0010.

Anti-inflammatory agents include Alclofenac; Alclometasone Dipropionate;Algestone Acetonide; α-Amylase; Amcinafal; Amcinafide; Amfenac Sodium;Amiprilose Hydrochloride; Anakinra; Anirolac; Anitrazafen; Apazone;Balsalazide Disodium; Bendazac; Benoxaprofen; Benzydamine Hydrochloride;Bromelains; Broperamole; Budesonide; Carprofen; Cicloprofen; Cintazone;Cliprofen; Clobetasol Propionate; Clobetasone Butyrate; Clopirac;Cloticasone Propionate; Cormethasone Acetate; Cortodoxone; Deflazacort;Desonide; Desoximetasone; Dexamethasone Dipropionate; DiclofenacPotassium; Diclofenac Sodium; Diflorasone Diacetate; Diflumidone Sodium;Diflunisal; Difluprednate; Diftalone; Dimethyl Sulfoxide; Drocinonide;Endrysone; Enlimomab; Enolicam Sodium; Epirizole; Etodolac; Etofenamate;Felbinac; Fenamole; Fenbufen; Fenclofenac; Fenclorac; Fendosal;Fenpipalone; Fentiazac; Flazalone; Fluazacort; Flufenamic Acid;Flumizole; Flunisolide Acetate; Flunixin; Flunixin Meglumine; FluocortinButyl; Fluorometholone Acetate; Fluquazone; Flurbiprofen; Fluretofen;Fluticasone Propionate; Furaprofen; Furobufen; Halcinonide; HalobetasolPropionate; Halopredone Acetate; Ibufenac; Ibuprofen; IbuprofenAluminum; Ibuprofen Piconol; Ilonidap; Indomethacin; IndomethacinSodium; Indoprofen; Indoxole; Intrazole; Isoflupredone Acetate;Isoxepac; Isoxicam; Ketoprofen; Lofemizole Hydrochloride; Lornoxicam;Loteprednol Etabonate; Meclofenamate Sodium; Meclofenamic Acid;Meclorisone Dibutyrate; Mefenamic Acid; Mesalamine; Meseclazone;Methylprednisolone Suleptanate; Morniflumate; Nabumetone; Naproxen;Naproxen Sodium; Naproxol; Nimazone; Olsalazine Sodium; Orgotein;Orpanoxin; Oxaprozin; Oxyphenbutazone; Paranyline Hydrochloride;Pentosan Polysulfate Sodium; Phenbutazone Sodium Glycerate; Pirfenidone;Piroxicam; Piroxicam Cinnamate; Piroxicam Olamine; Pirprofen;Prednazate; Prifelone; Prodolic Acid; Proquazone; Proxazole; ProxazoleCitrate; Rimexolone; Romazarit; Salcolex; Salnacedin; Salsalate;Salycilates; Sanguinarium Chloride; Seclazone; Sermetacin; Sudoxicam;Sulindac; Suprofen; Talmetacin; Talniflumate; Talosalate; Tebufelone;Tenidap; Tenidap Sodium; Tenoxicam; Tesicam; Tesimide; Tetrydamine;Tiopinac; Tixocortol Pivalate; Tolmetin; Tolmetin Sodium; Triclonide;Triflumidate; Zidometacin; Glucocorticoids; Zomepirac Sodium. Animportant anti-inflammatory agent is aspirin.

Other anti-inflammatory agents are cytokine inhibitors includingcytokine antagonists (e.g., IL-6 receptor antagonists), aza-alkyllysophospholipids (AALP), and Tumor Necrosis Factor-α (TNF-α)inhibitors, such as anti-TNF-α antibodies, soluble TNF receptor, TNF-α,antisense nucleic acid molecules, multivalent guanylhydrazone(CNI-1493), N-acetylcysteine, pentoxiphylline, oxpentifylline,carbocyclic nucleoside analogues, small molecule S9a, RP 55778 (a TNF-αsynthesis inhibitor), Dexanabinol (HU-211), MDL 201,449A(9-[(1R,3R)-trans-cyclopentan-3-ol]adenine, and trichodimerol(BMS-182123). Other TNF-α inhibitors include Etanercept (ENBREL,Immunex, Seattle) and Infliximab (REMICADE, Centocor, Malvern, Pa.).

Lipid reducing agents include gemfibrozil, cholystyramine, colestipol,nicotinic acid, and HMG-CoA reductase inhibitors. HMG-CoA reductaseinhibitors useful for administration, or co-administration with otheragents according to the invention include, but are not limited to,simvastatin (U.S. Pat. No. 4,444,784), lovastatin (U.S. Pat. No.4,231,938), pravastatin sodium (U.S. Pat. No. 4,346,227), fluvastatin(U.S. Pat. No. 4,739,073), atorvastatin (U.S. Pat. No. 5,273,995), andcerivastatin.

Calcium channel blockers include dihydropyridines, such as nifedipine,phenyl alkyl amines, such as verapamil, and benzothiazepines, such asdiltiazem. Other calcium channel blockers include, but are not limitedto, amrinone, amlodipine, bencyclane, felodipine, fendiline,flunarizine, isradipine, nicardipine, nimodipine, perhexilene,gallopamil, tiapamil and tiapamil analogues (such as 1993RO-11-2933),phenytoin, barbiturates, and the peptides dynorphin, omega-conotoxin,and omega-agatoxin, and the like and/or pharmaceutically acceptablesalts thereof

β-adrenergic receptor blocking agents include, but are not limited to,atenolol, acebutolol, alprenolol, befunolol, betaxolol, bunitrolol,carteolol, celiprolol, hedroxalol, indenolol, labetalol, levobunolol,mepindolol, methypranol, metindol, metoprolol, metrizoranolol,oxprenolol, pindolol, propranolol, practolol, practolol, sotalolnadolol,tiprenolol, tomalolol, timolol, bupranolol, penbutolol, trimepranol,2-(3-(1,1-dimethylethyl)-amino-2-hyd-roxypropoxy)-3-pyridenecarbonitrilHCl, 1-butylamino-3-(2,5-dichlorophenoxy-)-2-propanol,1-isopropylamino-3-(4-(2-cyclopropylmethoxyethyl)phenoxy)-2-propanol,3-isopropylamino-1-(7-methylindan-4-yloxy)-2-butanol,2-(3-t-butylamino-2-hydroxy-propylthio)-4-(5-carbamoyl-2-thienyl)thiazol,7-(2-hydroxy-3-t-butylaminpropoxy)phthalide. The above-identifiedcompounds can be used as isomeric mixtures, or in their respectivelevorotating or dextrorotating form.

A number of selective COX-2 inhibitors are known in the art and include,but are not limited to, COX-2 inhibitors described in U.S. Pat. No.5,474,995; U.S. Pat. No. 5,521,213; U.S. Pat. No. 5,536,752; U.S. Pat.No. 5,550,142; U.S. Pat. No. 5,552,422; U.S. Pat. No. 5,604,253; U.S.Pat. No. 5,604,260; U.S. Pat. No. 5,639,780; U.S. Pat. No. 5,677,318;U.S. Pat. No. 5,691,374; U.S. Pat. No. 5,698,584; U.S. Pat. No.5,710,140; U.S. Pat. No. 5,733,909; U.S. Pat. No. 5,789,413; U.S. Pat.No. 5,817,700; U.S. Pat. No. 5,849,943; U.S. Pat. No. 5,861,419; U.S.Pat. No. 5,922,742; U.S. Pat. No. 5,925,631; and U.S. Pat. No.5,643,933. A number of the above-identified COX-2 inhibitors areprodrugs of selective COX-2 inhibitors, and include those described inWO 95/00501, WO 95/18799, and U.S. Pat. No. 5,474,995, issued Dec. 12,1995.

Examples of angiotensin II antagonists include: peptidic compounds(e.g., saralasin, [(San1)(Val5)(Ala8)] angiotensin-(1-8) octapeptide andrelated analogs); N-substituted imidazole-2-one (U.S. Pat. No.5,087,634); imidazole acetate derivatives including2-N-butyl-4-chloro-1-(2-chlorobenzile) imidazole-5-acetic acid (see Longet al., J. Pharmacol. Exp. Ther. 247(1), 1-7 (1988));4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid andanalog derivatives (U.S. Pat. No. 4,816,463); N2-tetrazole β-glucuronideanalogs (U.S. Pat. No. 5,085,992); substituted pyrroles, pyrazoles, andtryazoles (U.S. Pat. No. 5,081,127); phenol and heterocyclic derivativessuch as 1,3-imidazoles (U.S. Pat. No. 5,073,566); imidazo-fused 7-memberring heterocycles (U.S. Pat. No. 5,064,825); peptides (e.g., U.S. Pat.No. 4,772,684); antibodies to angiotensin II (e.g., U.S. Pat. No.4,302,386); and aralkyl imidazole compounds such as biphenyl-methylsubstituted imidazoles (e.g., EP 253,310, Jan. 20, 1988); ES8891(N-morpholinoacetyl-(-1-naphthyl)-L-alany-1-(4, thiazolyl)-L-alanyl(35,45)-4-amino-3-hydroxy-5-cyclo-hexapentanoyl-N-hexylamide, SankyoCompany, Ltd., Tokyo, Japan); SKF108566 (E-α-2-[2-butyl-1-(carboxyphenyl)methyl]1H-imidazole-5-yl[methylane]-2-thiophenepropanoic acid,Smith Kline Beecham Pharmaceuticals, Pa.); Losartan (DUP753/MK954,DuPont Merck Pharmaceutical Company); Remikirin (RO42-5892, F. HoffmanLaRoche AG); A₂ agonists (Marion Merrill Dow) and certain non-peptideheterocycles (G. D. Searle and Company).

Angiotensin converting enzyme (ACE) inhibitors include amino acids andderivatives thereof, peptides, including di- and tri-peptides andantibodies to ACE which intervene in the renin-angiotensin system byinhibiting the activity of ACE thereby reducing or eliminating theformation of pressor substance angiotensin II. Other ACE inhibitorsinclude acylmercapto and mercaptoalkanoyl prolines such as captopril(U.S. Pat. No. 4,105,776) and zofenopril (U.S. Pat. No. 4,316,906),carboxyalkyl dipeptides such as enalapril (U.S. Pat. No. 4,374,829),lisinopril (U.S. Pat. No. 4,374,829), quinapril (U.S. Pat. No.4,344,949), ramipril (U.S. Pat. No. 4,587,258), and perindopril (U.S.Pat. No. 4,508,729), carboxyalkyl dipeptide mimics such as cilazapril(U.S. Pat. No. 4,512,924) and benazapril (U.S. Pat. No. 4,410,520),phosphinylalkanoyl prolines such as fosinopril (U.S. Pat. No. 4,337,201)and trandolopril.

Renin inhibitors include amino acids and derivatives thereof, peptidesand derivatives thereof, and antibodies to renin. Other rennininhibitors include urea derivatives of peptides (U.S. Pat. No.5,116,835); amino acids connected by nonpeptide bonds (U.S. Pat. No.5,114,937); di- and tri-peptide derivatives (U.S. Pat. No. 5,106,835);amino acids and derivatives thereof (U.S. Pat. Nos. 5,104,869 and5,095,119); diol sulfonamides and sulfinyls (U.S. Pat. No. 5,098,924);modified peptides (U.S. Pat. No. 5,095,006); peptidyl β-aminoacylaminodiol carbamates (U.S. Pat. No. 5,089,471); pyrolimidazolones (U.S.Pat. No. 5,075,451); fluorine and chlorine statine or statone containingpeptides (U.S. Pat. No. 5,066,643); peptidyl amino diols (U.S. Pat. Nos.5,063,208 and 4,845,079); N-morpholino derivatives (U.S. Pat. No.5,055,466); pepstatin derivatives (U.S. Pat. No. 4,980,283);N-heterocyclic alcohols (U.S. Pat. No. 4,885,292); monoclonal antibodiesto renin (U.S. Pat. No. 4,780,401); and a variety of other peptides andanalogs thereof (U.S. Pat. Nos. 5,071,837, 5,064,965, 5,063,207,5,036,054, 5,036,053, 5,034,512, and 4,894,437).

Other therapeutic agents useful in treating diabetes and relateddisorders include, but are not limited to, lipase inhibitors such ascetilistat (ATL-962); synthetic amylin analogs such as Symlinpramlintide with or without recombinant leptin; sodium-glucosecotransporter 2 (SGLT2) inhibitors like sergliflozin (869682; KGT-1251),YM543, canagliflozin, ertugliflozin, dapagliflozin, GlaxoSmithKlinemolecule 189075, and Sanofi-Aventis molecule AVE2268; dual adiposetriglyceride lipase and PI3 kinase activators like Adyvia (ID 1101);antagonists of neuropeptide Y2, Y4, and Y5 receptors like Nastechmolecule PYY3-36, synthetic analog of human hormones PYY3-36 andpancreatic polypeptide (7TM molecule TM30338); Shionogi molecule S-2367;cannabinoid CB1 receptor antagonists such as rimonabant (Acomplia),taranabant, CP-945,598, Solvay molecule SLV319, Vernalis moleculeV24343; hormones like oleoyl-estrone; inhibitors of serotonin, dopamine,and norepinephrine (also known in the art as triple monoamine reuptakeinhibitors) like tesofensine (Neurosearch molecule NS2330); inhibitorsof norepinephrine and dopamine reuptake, like Contrave (bupropion plusopioid antagonist naltrexone) and Excalia (bupropion plus anticonvulsantzonisaminde); inhibitors of 11β-hydroxysteroid dehydrogenase type 1(11b-HSD1) like Incyte molecule INCB13739; inhibitors of cortisolsynthesis such as ketoconazole (DiObex molecule DIO-902); inhibitors ofgluconeogenesis such as Metabasis/Daiichi molecule CS-917; glucokinaseactivators like Roche molecule R1440; antisense inhibitors of proteintyrosine phosphatase-1B such as ISIS 113715; as well as other agentslike NicOx molecule NCX 4016; injections of gastrin and epidermal growthfactor (EGF) analogs such as Islet Neogenesis Therapy (E1-I.N.T.);betahistine (Obecure molecule OBE101); bile acid sequestrants (e.g.,cholestyramine and colestipol), vitamin B₃ (also known as nicotinicacid, or niacin), vitamin B₆ (pyridoxine), vitamin B₁₂ (cyanocobalamin),flbric acid derivatives (e.g., gemfibrozil, clofibrate, fenofibrate andbenzaflbrate), probucol, nitroglycerin, and inhibitors of cholesterolabsorption (e.g., β-sitosterol and acylCoA-cholesterol acyltransferase(ACAT) inhibitors such as melinamide), HMG-CoA synthase inhibitors,squalene epoxidase inhibitors and squalene synthetase inhibitors.

Examples of agents used to treat Cushing's syndrome include, withoutlimitation, neuromodulators (Signifor® (pasireotide), cabergoline);adrenal steroidogenesis inhibitors (ketoconazole, metyrapone, mitotane,etomidate); and nuclear receptor modulators (Korlym® (mifepristone),retinoic acid). Other agents include, without limitation, epidermalgrowth factor receptor inhibitors (for example, gefitinib), thealdosterone synthase/11β-hydroxylase inhibitor LCI699, andlevoketoconazole (COR-003).

Examples of analgesic agents frequently used to treat pain, includingneuropathic pain, include, without limitation, opioid or non-opioidanalgesic agents. Suitable opioid analgesic agents include, but are notlimited to, morphine, heroin, hydromorphone, hydrocodone, oxymorphone,oxycodone, metopon, apomorphine, normorphine, etorphine, buprenorphine,meperidine, lopermide, anileridine, ethoheptazine, piminidine,betaprodine, diphenoxylate, fentanil, sufentanil, alfentanil,remifentanil, levorphanol, dextromethorphan, phenazocine, pentazocine,cyclazocine, methadone, isomethadone and propoxyphene. Suitablenon-opioid analgesic agents include, but are not limited to, aspirin,celecoxib, rofecoxib, diclofinac, diflusinal, etodolac, fenoprofen,flurbiprofen, ibuprofen, ketoprofen, indomethacin, ketorolac,meclofenamate, mefanamic acid, nabumetone, naproxen, piroxicam andsulindac.

Examples of therapeutic agents frequently used to treat glaucoma includecholinergic agonists (e.g., pilocarpine and carbachol), cholinesteraseinhibitors (e.g., physostigmine, neostigmine, demacarium, echothiophateiodide and isofluorophate), carbonic anhydrase inhibitors (e.g.,acetazolamide, dichlorphenamide, methazolamide, ethoxzolamide anddorzolamide), non-selective adrenergic agonists (e.g., epinephrine anddipivefrin), a₂-selecteive adrenergic agonists (e.g., apraclonidine andbrimonidine), β-blockers (e.g., timolol, betazolol, levobunolol,carteolol and metipranolol), prostaglandin analogs (e.g., latanoprost)and osmotic diuretics (e.g., glycerin, mannitol and isosorbide);corticosteroids, such as beclomethasone, methylprednisolone,betamethasone, prednisone, prenisolone, dexamethasone, fluticasone andhydrocortisone, and corticosteroid analogs such as budesonide.

Examples of therapeutic agents frequently used to treat Alzheimer'sdisease include β-secretase inhibitors or γ-secretase inhibitors;glycine transport inhibitors, tau phosphorylation inhibitors; blockersof Aβ oligomer formation; p25/CDK5 inhibitors; HMG-CoA reductaseinhibitors; PPARγ agonists, such as pioglitazone and rosiglitazone;NK1/NK3 receptor antagonists; NSAID's including ibuprofen; vitamin E;anti-amyloid antibodies, including anti-amyloid humanized monoclonalantibodies; COX-2 inhibitors; anti-inflammatory compounds, such as(R)-flurbiprofen; CB-1 receptor antagonists or CB-1 receptor inverseagonists; antibiotics such as doxycycline and rifampin;N-methyl-D-aspartate (NMDA) receptor antagonists, such as memantine andneramexane; NR2B antagonists; androgen receptor modulators;acetylcholinesterase inhibitors such as galantamine, rivastigmine,donepezil, and tacrine; mGluR5 modulators; growth hormone secretagoguessuch as ibutamoren, ibutamoren mesylate, and capromorelin; histamine H₃antagonists; AMPA agonists; PDE IV inhibitors; GABA_(A) inverseagonists; GABA_(A) α5 receptor ligands; GABA_(B) receptor ligands;potassium channel blockers; neuronal nicotinic agonists; P-450inhibitors, such as ritonavir.

Examples of therapeutic agents frequently used to treat affectivedisorders such as depression include, without limitation, amitriptyline,amitriptyline oxide, desipramine, dibenzepin, dosulepin, doxepin,chloroimipramine, imipramine, nortriptyline, mianserin, maprotiline,trimipramine, CP-122721, elzasonan, PD-171729, MK-869, DOV-216303,DOV-21947, licarbazepine, amfebutamone, radafaxine, vilazodone,GSK-679769, GW-597599, NS-2359, GSK-876008, pramipexole, duloxetine,atomoxetine, LY-628535, desvenlafaxine, escitalopram, LU-AA21004,saredutant, SR-58611, SSR-149415, SSR-146977, moclobemide, R-673,R-1204, BMS-469458, DPC-368, Org-34517, Org-34850, inhibitors of the CRHreceptors, ONO-2333Ms, NBI-876008, AAG-561, NBI-34041, DPC-368,PD-171729, SSR-125543, viloxazine, trazodone, nefazodone, mirtazapine,venlafaxine, reboxetine, tranylcypromine, brofaromine, moclobemide,citalopram, escitalopram, paroxetine, fluoxetine, fluvoxamine,sertraline, Hypericum (St. John's Wort), alprazolam, clonazepam,diazepam, lorazepam, halazepam, chlordiazepoxide, and other drugs suchas buspirone, clonidine, pagoclone, risperidone, olanzapine, quetiapine,ziprasidone, celecoxib, piroxicam, parecoxib, valdecoxib, PMI-001,PH-686464, SC-58236, etoricoxib, rofecoxib, L-776967, lumiracoxib,GW-406381, GW-644784, meloxicam, SVT-2016, PAC-10649, CS-706, LAS-34475,cimicoxib, A-183827.0, or nimesulide.

Examples of therapeutic agents frequently used to treat addiction anddrug abuse include, without limitation, phenelzine, phenylalkylhydrazine(U.S. Pat. No. 4,786,653), disulfiram (“Antabuse”),2-imino-5-phenyl-4-oxazolidinone, α-methyl-para-tyrosine or fusaricacid, piperazine derivatives (U.S. Pat. No. 4,325,952), clonidine inconjunction with a tricyclic antidepressant drug (U.S. Pat. No.4,788,189), γ-pyrones such as maltol or ethyl maltol (U.S. Pat. No.4,276,890), acamprosate, gabapentin, vigabatrin, baclofen,N-acetylcysteine, nocaine, modanafil, paroxetine, bupropion,mirtazapine, topiramate, ondansetron, varenicline, antagonists of opioidreceptors such as naltrexone, naloxone, nalmephine, antaxone, L-α-acetylmethadol, pentazocine, butorphanol, nalbuphine, buprenorphine, andmethadone.

Examples of therapeutic agents frequently used in osteoporosistreatments, and may modulate bone mineral content include, but are notlimited to, bisphosphonates such as alendronate (Fosamax®), risedronate(Actonel®), etidronate (Didronel®), pamidronate, tiludronate (Skelid®),clodronate (Bonefos®; Loron®), neridronate, olpadronate, zoledronate(Zometa®), and ibandronate (Boniva®), selective estrogen-receptormodulators (SERMs) such as raloxifene (Evista®), arzoxifene, clomifene,bazedoxifene, lasofoxifene, ormeloxifene, tamoxifen, and toremifine,anabolic therapies such as teriparatide (Forteo®; recombinantparathyroid hormone), and strontium ranelate, and recombinant peptidefragments of parathyroid hormone, estrogen/progesterone replacementtherapies, monoclonal antibodies, inhibitors of receptor activator ofnuclear factor kB ligand (RANKL) such as denosumab, osteoprotegerin andPepstatin A, inhibitors of cathepsin K such as but not limited toOST-4077 (furan-2-carboxylicacid-(1-{1-[4-fluoro-2-(2-oxo-pyrrolidin-1-yl)-phenyl]-3-oxo-piperidin-3-ylcarbamoyl}-cyclohexyl)-amide),leupeptin, Cbz-Phe-Ala-CHN2, Cbz-Leu-Leu-Leu-aldehyde, cystatin,irreversible cysteine protease inhibitors like peptidehalomethylketones, peptide diazomethylketones, and epoxides, quiescentirreversible cysteine protease inhibitors such as acyloxymethylketones,azapeptides, Michael acceptors like peptide vinyl esters, sulfones andsulfonates, reversible cysteine protease inhibitors such as peptidealdehydes, a-ketoesters and a-ketoamides, peptide methyl ketones andhydroxyl, alkyloxy, aryloxy, alkylthio, and arylthio derivativesthereof, 1,3-bis-(acylamino)-2-propanones,1,3-bis-(acylhydrazino)-carbonyls, acylamino-pyrazolones, piperidinones,and thiazone-carbonyl-hydrazides, antagonists of integrin Avb3 (alsoknown in the art as vitronectin), calcilytic compounds (Ca2+ receptorantagonists which increase the secretion of PTH), calcitonin(MiacalcinÒ), nitrates including but not limited to isosorbidemononitrate (ISMO) or nitroglycerin ointment (NTG), and dietarysupplements such as calcium and vitamin D, and combinations thereof.

Another embodiment of the present invention is a method of identifyingpatients in need of treatment based on measuring clock gene (e.g. Cry1and Cry2) expression levels in samples taken from a subject (Bjarnason,G. A. et al. Am. J. Pathol. 2001, 158, 1793; Akashi, M. et. al. Proc.Natl. Acad. Sci. USA, 2010, 107, 15643). Rhythmic mRNA expressionprofiles for human clock genes, including Cry1 and Cry2, measured insamples from a subject indicate a circadian clock is present inperipheral tissues (Mohawk, J. A. et al. Ann. Rev. Neurosci. 2012, Epubahead of print). Expression of circadian clock related genes in thesesamples has been demonstrated to vary during the day. Furthermore, clockgene (e.g. Cry1 and Cry2) expression patterns in peripheral bloodmononuclear cells are altered in humans by diseases such as obesity(Tahira, K. et al. Arch. Med. Sci. 2011, 7, 933). Changes in clock gene(e.g. Cry1 and Cry2) expression in peripheral mononuclear blood cellscan be correlated with serum leptin, adiponectin, insulin and hsCRPlevels, plasma lipid, glucose, melatonin and cortisol levels and, inanimals, expression of clock genes (e.g. Cry1 and Cry2) in tissuesincluding liver, adipose, pancreas and skeletal muscle. By contactingsamples taken from a subject treated with a compound of formula I andmeasuring rhythmic mRNA or protein expression profiles, patients in needof treatment may be identified and pharmacological activity can beassessed. In other embodiments, the activities of one or morecryptochromes may be assessed, for example, the ability of cryptochromesto bind to a target such as Per1, Per2, glucocorticoid receptor (GR), ora promoter sequence containing Cry recognition sites, such as, e.g., theCLOCK-BMAL1 promoter.

Accordingly, one aspect of the subject matter disclosed herein relatesto a method of monitoring progression or prognosis of a Cry-mediateddisease or disorder in a subject, comprising measuring an effectiveamount of one or more cryptochromes in a first sample from the subjectat a first period of time; measuring an effective amount of one or morecryptochromes in a second sample from the subject at a second period oftime; and comparing the amount of the one or more cryptochromes detectedin the first sample to the amount of the one or more cryptochromesdetected in the second sample, or to a reference value.

“Diagnosis”, “diagnose”, “prognose” or “prognosis” is not limited to adefinitive or near definitive determination that an individual has adisease, but also includes determining that an individual has anincreased likelihood of having or developing the disease, compared tohealthy individuals or to the general population.

As used herein, “expression” and “expression levels” include but are notlimited to one or more of the following: transcription of the gene intoprecursor mRNA; splicing and other processing of the precursor mRNA toproduce mature mRNA; mRNA stability; translation of the mature mRNA intoprotein (including codon usage and tRNA availability); and glycosylationand/or other modifications of the translation product, if required forproper expression and function.

A “formula,” “algorithm,” or “model” is any mathematical equation,algorithmic, analytical or programmed process, or statistical techniquethat takes one or more continuous or categorical inputs (herein called“parameters”) and calculates an output value, sometimes referred to asan “index” or “index value.” Non-limiting examples of “algorithms”include sums, ratios, and regression operators, such as coefficients orexponents, value transformations and normalizations (including, withoutlimitation, those normalization schemes based on clinical parameters,such as gender, age, body mass index, or ethnicity), rules andguidelines, statistical classification models, and neural networkstrained on historical populations. Of particular use in measuring Cry asdefined herein are linear and non-linear equations and statisticalclassification analyses to “correlate” the relationship between levelsof Cry detected in a subject sample and the subject's risk of developinga Cry-mediated disease or disorder.

“Measuring” or “measurement” means assessing the presence, absence,quantity or amount (which can be an effective amount) of either a givensubstance within a clinical or subject-derived sample, including thederivation of qualitative or quantitative concentration levels of suchsubstances, or otherwise evaluating the values or categorization of asubject's clinical parameters. Measurement or measuring may also involvequalifying the type or identifying the substance. Measurement ormeasuring may also involve the ability of one or more Cry to bind to atarget, wherein the target may be period genes or proteins Per1 andPer2, glucocorticoid receptor (GR), or the promoter region of theCLOCK-BMAL1 gene. Measurement of Cry may be used to diagnose, detect, oridentify a Cry-mediated disease or disorder in a subject, to monitor theprogression or prognosis of a Cry-mediated disease or disorder in asubject, to predict the recurrence of a Cry-mediated disease or disorderin a subject, or to classify a subject as having a low risk or a highrisk of developing a Cry-mediated disease or disorder or a recurrence ofa Cry-mediated disease or disorder.

“Risk” in the context of the present invention relates to theprobability that an event will occur over a specific time period, as inthe development of Cry-mediated disease or disorder, and can mean asubject's “absolute” risk or “relative” risk. Absolute risk can bemeasured with reference to either actual observation post-measurementfor the relevant time cohort, or with reference to index valuesdeveloped from statistically valid historical cohorts that have beenfollowed for the relevant time period. Relative risk refers to the ratioof absolute risks of a subject compared either to the absolute risks oflow risk cohorts or an average population risk, which can vary by howclinical risk factors are assessed. Odds ratios, the proportion ofpositive events to negative events for a given test result, are alsocommonly used (odds are according to the formula p/(1−p) where p is theprobability of event and (1−p) is the probability of no event) tono-conversion. Alternative continuous measures which may be assessed inthe context of the present invention include time to development of aCry-mediated disease or disorder, or progression to a different stage ofa Cry-mediated disease or disorder, including progression or developmentof a Cry-mediated disease or disorder and therapeutic conversion riskreduction ratios.

“Risk evaluation,” or “evaluation of risk” in the context of the subjectmatter disclosed herein encompasses making a prediction of theprobability, odds, or likelihood that an event or disease state mayoccur, the rate of occurrence of the event or conversion from onedisease state to another, i.e., from a “normal” condition to an at-riskcondition for developing a Cry-mediated disease or disorder, or from anat-risk condition to a Cry-mediated disease or disorder, or developmentof recurrent disease or disorder. Risk evaluation can also compriseprediction of other indices of Cry-mediated disease or disorder, eitherin absolute or relative terms in reference to a previously measuredpopulation. The methods of the present invention may be used to makecontinuous or categorical measurements of the risk of conversion to aCry-mediated disease or disorder, thus diagnosing and defining the riskspectrum of a category of subjects defined as at risk for developing thedisease or disorder. In the categorical scenario, the invention can beused to discriminate between normal and at-risk subject cohorts. Inother embodiments, the present invention may be used so as todiscriminate at-risk conditions from disease conditions, or diseaseconditions from normal.

A “sample” as used herein is a biological sample isolated from a subjectand can include, by way of example and not limitation, whole blood,serum, plasma, blood cells, endothelial cells, tissue biopsies,lymphatic fluid, ascites fluid, interstitial fluid (also known as“extracellular fluid” and encompasses the fluid found in spaces betweencells, including, inter alia, gingival crevicular fluid), bone marrow,seminal fluid, cerebrospinal fluid (CSF), saliva, mucous, sputum, sweat,urine, or any other secretion, excretion, or other bodily fluids.

By “statistically significant”, it is meant that the alteration isgreater than what might be expected to happen by chance alone (whichcould be a “false positive”). Statistical significance can be determinedby any method known in the art. Commonly used measures of significanceinclude the p-value, which presents the probability of obtaining aresult at least as extreme as a given data point, assuming the datapoint was the result of chance alone. A result is often consideredhighly significant at a p-value of 0.05 or less.

The risk of a Cry-mediated disease or disorder can be detected bymeasuring an “effective amount” of one or more cryptochromes in a sample(e.g., a subject derived sample), and comparing the effective amounts toreference values, often utilizing mathematical algorithms or formulae inorder to combine information from results of multiple individuals into asingle measurement. Subjects identified as having an increased risk of aCry-mediated disease or disorder can optionally be selected to receivetreatment regimens or therapeutic interventions, such as administrationof the compounds of formula I as defined herein as monotherapy or incombination with one or more additional therapeutic agents, orimplementation of surgical interventions (which may follow or precedeadministration of the compounds of formula I, alone or in combinationwith additional therapeutic agents or other therapies).

The methods for detecting these cryptochromes in a sample have manyapplications. For example, one or more cryptochromes can be measured toaid diagnosis or prognosis of a Cry-mediated disease or disorder. Inanother example, the methods for detection of the cryptochromes can beused to monitor responses in a subject to treatment of a Cry-mediateddisease or disorder. In another example, the methods can be used toassay for and to identify compounds that modulate expression ofcryptochromes in vivo or in vitro.

The present invention may be used to make continuous or categoricalmeasurements of the risk of conversion to a Cry-mediated disease ordisorder, thus diagnosing and defining the risk spectrum of a categoryof subjects defined as being at-risk for developing the disease ordisorder. In the categorical scenario, the methods of the presentinvention can be used to discriminate between normal and at-risk subjectcohorts. In other embodiments, the present invention may be used so asto discriminate at-risk from disease, or disease from normal. Suchdiffering use may require different combinations in individual panel orprofile, mathematical algorithm, and/or cut-off points, but be subjectto the same aforementioned measurements of accuracy for the intendeduse.

Identifying the at-risk subject enables the selection and initiation ofvarious therapeutic interventions or treatment regimens in order todelay, reduce, or prevent that subject's conversion to a Cry-mediateddisease or disorder. Levels of an effective amount of cryptochromeproteins, nucleic acids, polymorphisms, metabolites, or other analytesalso allows for the course of treatment to be monitored. In this method,a biological sample can be provided from a subject undergoing treatmentregimens, e.g., therapeutic treatments, for a Cry-mediated disease ordisorder. Such treatment regimens can include, but are not limited to,surgical intervention and treatment with therapeutic agents used insubjects diagnosed or identified with a Cry-mediated disease ordisorder, for example, the compounds of formula I described herein. Ifdesired, biological samples are obtained from the subject at varioustime points before, during, or after treatment. For example, determiningthe disease status by comparison of a subject's cryptochrome profile toa reference cryptochrome profile can be repeated more than once, whereinthe subject's profile can be obtained from a separate sample taken eachtime the method is repeated. Samples may be taken from the subject atdefined time intervals, such as, e.g., 4 hours, 8 hours, 12 hours, 24hours, 48 hours, 72 hours, or any suitable time interval as would beperformed by those skilled in the art.

Differences in the genetic makeup of subjects can result in differencesin their relative abilities to metabolize various drugs, which maymodulate the symptoms or risk factors of a Cry-mediated disease ordisorder. Subjects that have a Cry-mediated disease or disorder, or areat risk for developing a Cry-mediated disease or disorder can vary inage, ethnicity, and other parameters. Accordingly, measuring effectiveamounts of one or more cryptochromes as defined herein, both alone andtogether in combination with known genetic factors for drug metabolism,allow for a pre-determined level of predictability that a putativetherapeutic or prophylactic to be tested in a selected subject will besuitable for treating or preventing a Cry-mediated disease or disorderin the subject.

To identify therapeutic agents or drugs that are appropriate for aspecific subject, a test sample from the subject can also be exposed toa therapeutic agent or a drug, and the level or activity of one or moreof cryptochrome proteins, nucleic acids, polymorphisms, splice variants,metabolites or other analytes can be determined. Other genes or proteinsthat are affected or which directly or indirectly bind to one or morecryptochromes (e.g., Per1, Per2, GR, CLOCK-BMAL1 promoter, etc.) mayalso be measured. The level of one or more cryptochromes can be comparedto sample derived from the subject before and after subject managementfor a Cry-mediated disease or disorder, e.g., treatment or exposure to atherapeutic agent or a drug, or can be compared to samples derived fromone or more subjects who have shown improvements in risk factors as aresult of such treatment or exposure.

Nucleic acids may be obtained from the samples in many ways known to oneof skill in the art, for example, extraction methods, including e.g.,solvent extraction, affinity purification and centrifugation. Selectiveprecipitation can also purify nucleic acids. Chromatography methods mayalso be utilized including, gel filtration, ion exchange, selectiveadsorption, or affinity binding. The nucleic acids may be, for example,RNA, DNA or may be synthesized into cDNA. The nucleic acids may bedetected using microarray techniques that are well known in the art, forexample, Affymetrix arrays followed by multidimensional scalingtechniques. See R. Ekins, R. and Chu, F. W. (1999) Trends Biotechnol.17: 217-218; D. D. Shoemaker, et al., (2001) Nature 409(6822): 922-927and U.S. Pat. No. 5,750,015.

If desired, the sample can be prepared to enhance detectability of oneor more cryptochromes by, for example, pre-fractionation. Methods ofpre-fractionation include, for example, Cibacron blue agarosechromatography, size exclusion chromatography, ion exchangechromatography, heparin chromatography, lectin chromatography, affinitychromatography, single stranded DNA affinity chromatography, sequentialextraction, gel electrophoresis and liquid chromatography. The analytesalso may be modified prior to detection. A sample can bepre-fractionated by removing proteins that are present in a highquantity or that may interfere with the detection of molecules ofinterest in a sample. For example, in a blood serum sample, serumalbumin is present in a high quantity and may obscure the analysis ofone or more cryptochromes. Thus, a blood serum sample can bepre-fractionated by removing serum albumin using, for example, asubstrate that comprises adsorbents that specifically bind serumalbumin, an affinity column or anti-serum albumin antibodies can beused.

In other embodiments, molecules of interest in a sample can be separatedby high-resolution electrophoresis, e.g., one or two-dimensional gelelectrophoresis. A fraction can be isolated and further analyzed by gasphase ion spectrometry. Preferably, two-dimensional gel electrophoresisis used to generate two-dimensional array of spots, including one ormore cryptochromes. See, e.g., Jungblut and Thiede, (1997) Mass Spectr.Rev. 16: 145-162. The two-dimensional gel electrophoresis can beperformed using methods known in the art. See, e.g., Deutscher ed.,Methods in Enzymology vol. 182. Typically, a sample may be separated by,e.g., isoelectric focusing, during which one or more cryptochromes in asample are separated in a pH gradient until they reach a spot wheretheir net charge is zero (i.e., isoelectric point). This firstseparation step results in one-dimensional array. The molecules inone-dimensional array are further separated using a technique generallydistinct from that used in the first separation step. For example, inthe second dimension, molecules of interest separated by isoelectricfocusing are further separated using a polyacrylamide gel, such aspolyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate (SDS-PAGE). SDS-PAGE gel allows further separation based onmolecular mass. Typically, two-dimensional gel electrophoresis canseparate chemically different molecules of interest in the molecularmass range from 1000-200,000 Da within complex mixtures.

Molecules of interest in the two-dimensional array can be detected usingany suitable methods known in the art. For example, molecules ofinterest in a gel can be labeled or stained (e.g., Coomassie Blue orsilver staining) If gel electrophoresis generates spots that correspondto the molecular weight of one or more cryptochromes of the invention,the spot can be excised and further analyzed by, for example, gas phaseion spectrometry, mass spectrometry, or high performance liquidchromatography. Alternatively, the gel containing molecules of interestcan be transferred to an inert membrane by applying an electric field.Then a spot on the membrane that approximately corresponds to themolecular weight of a molecule of interest can be analyzed by e.g., gasphase ion spectrometry, mass spectrometry, or HPLC.

Optionally, a molecule of interest can be modified before analysis toimprove its resolution or to determine its identity. For example, thesample may be subject to proteolytic digestion before analysis. Anyprotease can be used. Proteases, such as trypsin, that are likely tocleave proteins into a discrete number of fragments are particularlyuseful. The fragments that result from digestion may function as afingerprint for the molecules of interest, thereby enabling theirindirect detection. This is particularly useful where there aremolecules of interest with similar molecular masses that might beconfused for the preferred molecule, i.e., cryptochromes, in question.Also, proteolytic fragmentation is useful for high molecular weightmolecules because smaller molecules are more easily resolved by massspectrometry. In another example, molecules can be modified to improvedetection resolution. For instance, neuraminidase can be used to removeterminal sialic acid residues from glycoproteins to improve binding toan anionic adsorbent (e.g., cationic exchange arrays) and to improvedetection resolution. In another example, the molecules can be modifiedby the attachment of a tag of particular molecular weight thatspecifically binds to another molecular entity, further distinguishingthem. Optionally, after detecting such modified molecules of interest,the identity of the molecules can be further determined by matching thephysical and chemical characteristics of the modified versions in aprotein database (e.g., SwissProt).

Once captured on a substrate, e.g., biochip or antibody, any suitablemethod, such as those described herein as well as other methods known inthe art, can be used to measure one or more cryptochromes in a sample.The actual measurement of levels or amounts of the such molecules can bedetermined using any method known in the art. These methods include,without limitation, mass spectrometry (e.g., laser desorption/ionizationmass spectrometry), fluorescence (e.g. sandwich immunoassay), surfaceplasmon resonance, ellipsometry and atomic force microscopy. Methods mayfurther include, by one or more of microarrays, PCR methods, massspectrometry (including, for example, and without limitation, ESI-MS,ESI-MS/MS, ESI-MS/(MS)n, matrix-assisted laser desorption ionizationtime-of-flight mass spectrometry (MALDI-TOF-MS), surface-enhanced laserdesorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS),desorption/ionization on silicon (DIOS), secondary ion mass spectrometry(SIMS), quadrupole time-of-flight (Q-TOF), atmospheric pressure chemicalionization mass spectrometry (APCI-MS), APCI-MS/MS, APCI-(MS)n,atmospheric pressure photoionization mass spectrometry (APPI-MS),APPI-MS/MS, and APPI-(MS)n, quadrupole mass spectrometry, Fouriertransform mass spectrometry (FTMS), and ion trap mass spectrometry),nucleic acid chips, Northern blot hybridization, TMA, SDA, NASBA, PCR,real time PCR, reverse transcriptase PCR, real time reversetranscriptase PCR, in situ PCR, chromatographic separation coupled withmass spectrometry, protein capture using immobilized antibodies or bytraditional immunoassays. See for example, U.S. Pat. Nos. 5,723,591;5,801,155 and 6,084,102 and Higuchi, 1992 and 1993. PCR assays may bedone, for example, in a multi-well plate formats or in chips, such asthe BioTrove OPEN ARRAY Chips (BioTrove, Woburn, Mass.).

For example, sequences within the sequence database entriescorresponding to cryptochromes can be used to construct probes fordetecting RNA sequences in, e.g., Northern blot hybridization analysesor methods which specifically, and, preferably, quantitatively amplifyspecific nucleic acid sequences. As another example, the sequences canbe used to construct primers which specifically or selectively hybridizeto cryptochrome sequences and which are used to amplifying suchsequences in, e.g., amplification-based detection methods such asreverse-transcription based polymerase chain reaction (RT-PCR), e.g.,quantitative real-time RT-PCR. When alterations in gene expression areassociated with gene amplification, deletion, polymorphisms, andmutations, sequence comparisons in test and reference populations can bemade by comparing relative amounts of the examined DNA sequences insubject and reference cell populations. As used herein, the term“specifically (or selectively) hybridizes” when referring to a nucleicacid, refers to a binding reaction that is determinative of the presenceof the nucleic acid in a heterogeneous population of nucleic acids.Thus, under designated assay conditions, the specified nucleic acidprobe (including inhibitory nucleic acids) may bind or hybridize to aparticular nucleic acid of interest at least two times the backgroundand do not substantially bind or hybridize in a significant amount toother nucleic acids present in the sample.

Levels of cryptochromes can also be determined by immunoassay. Theantibody may be monoclonal, polyclonal, chimeric, or a fragment of theforegoing, as discussed in detail herein, and the step of detecting thereaction product may be carried out with any suitable immunoassay. Thephrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein in a heterogeneous population of proteinsand other biologies. Thus, under designated immunoassay conditions, thespecified antibodies bind to a particular protein at least two times thebackground and do not substantially bind in a significant amount toother proteins present in the sample. Specific binding to an antibodyunder such conditions may require an antibody that is selected for itsspecificity for a particular protein. For example, polyclonal antibodiesraised to a cryptochrome from specific species such as rat, mouse, orhuman can be selected to obtain only those polyclonal antibodies thatare specifically immunoreactive with that cryptochrome and not withother proteins, except for polymorphic variants and alleles of thecryptochrome. This selection may be achieved by subtracting outantibodies that cross-react with cryptochromes from other species.

Immunoassays carried out in accordance with the present invention may behomogeneous assays or heterogeneous assays. In a homogeneous assay theimmunological reaction usually involves the specific antibody (e.g.,anti-cryptochrome protein antibody), a labeled analyte, and the sampleof interest. The signal arising from the label is modified, directly orindirectly, upon the binding of the antibody to the labeled analyte.Both the immunological reaction and detection of the extent thereof canbe carried out in a homogeneous solution. Immunochemical labels whichmay be employed include free radicals, radioisotopes, fluorescent dyes,enzymes, bacteriophages, or coenzymes.

In a heterogeneous assay approach, the reagents are usually the sample,the antibody, and means for producing a detectable signal. Samples asdescribed above may be used. The antibody can be immobilized on asupport, such as a bead (such as protein A and protein G agarose beads),plate or slide, and contacted with the specimen suspected of containingthe antigen in a liquid phase. The support is then separated from theliquid phase and either the support phase or the liquid phase isexamined for a detectable signal employing means for producing suchsignal. The signal is related to the presence of the analyte in thesample. Means for producing a detectable signal include the use ofdetectable labels. Exemplary detectable labels include magnetic beads(e.g., DYNABEADS™), fluorescent dyes, enzymes (e.g., horse radishperoxide, alkaline phosphatase and others commonly used in an ELISA),radiolabels (e.g., ³⁵S, ¹²⁵I, ¹³¹I), and fluorescent labels (e.g.,fluorescein, Alexa, green fluorescent protein, rhodamine) andcolorimetric labels such as colloidal gold or colored glass or plasticbeads in accordance with known techniques.

Alternatively, the molecule of interest in the sample can be detectedusing an indirect assay, wherein, for example, a second, labeledantibody is used to detect bound cryptochrome-specific antibody, and/orin a competition or inhibition assay wherein, for example, a monoclonalantibody which binds to a distinct epitope of the cryptochrome isincubated simultaneously with the mixture. For example, if the antigento be detected contains a second binding site, an antibody which bindsto that site can be conjugated to a detectable group and added to theliquid phase reaction solution before the separation step. The presenceof the detectable label on the solid support indicates the presence ofthe antigen in the test sample. Methods for measuring the amount or thepresence of antibody-antigen complexes include, for example, detectionof fluorescence, luminescence, chemiluminescence, absorbance,reflectance, transmittance, birefringence or refractive index (e.g.,surface plasmon resonance, ellipsometry, a resonant mirror method, agrating coupler waveguide method or interferometry). Optical methodsinclude microscopy (both confocal and non-confocal), imaging methods andnon-imaging methods. Electrochemical methods include voltammetry andamperometry methods. Radio frequency methods include multipolarresonance spectroscopy. Examples of suitable immunoassays include, butare not limited to immunoblotting (e.g., Western blotting, slot blotassay), immunoprecipitation, immunofluorescence methods,chemiluminescence methods, electrochemiluminescence (ECL) orenzyme-linked immunoassays, e.g., enzyme-linked immunosorbent assay(ELISA) and radioimmunoassay (RIA). See generally E. Maggio,Enzyme-Immunoassay, (1980) (CRC Press, Inc., Boca Raton, Fla.); see alsoU.S. Pat. Nos. 4,727,022; 4,659,678; 4,376,110; 4,275,149; 4,233,402;and 4,230,767. These methods are also described in, e.g., Methods inCell Biology: Antibodies in Cell Biology, volume 37 (Asai, ed. 1993);Basic and Clinical Immunology (Stites & Ten, eds., 7th ed. 1991); andHarlow & Lane, supra. All of these are incorporated by reference herein.

Immunoassays can be used to determine presence or absence of one or morecryptochromes in a sample as well as the quantity in a sample. Theamount of an antibody-marker complex can be determined by comparing to astandard. A standard can be, e.g., a known compound or another proteinknown to be present in a sample. As noted above, the test amount of theone or more cryptochromes need not be measured in absolute units, aslong as the unit of measurement can be compared to a control.

Proteins frequently exist in a sample in a plurality of different formscharacterized by a detectably different mass. These forms can resultfrom either, or both, of pre- and post-translational modification.Pre-translational modified forms include allelic variants, slicevariants and RNA editing forms. Post-translationally modified formsinclude forms resulting from proteolytic cleavage (e.g., fragments of aparent protein), glycosylation, phosphorylation, lipidation, oxidation,methylation, cystinylation, sulphonation and acetylation. Antibodies canalso be useful for detecting post-translational modifications ofproteins, polypeptides, mutations, and polymorphisms, such as tyrosinephosphorylation, threonine phosphorylation, serine phosphorylation,glycosylation (e.g., O-GlcNAc). Such antibodies specifically detect thephosphorylated amino acids in a protein or proteins of interest, and canbe used in immunoblotting, immunofluorescence, and ELISA assaysdescribed herein. These antibodies are well-known to those skilled inthe art, and commercially available. Post-translational modificationscan also be determined using metastable ions in reflectormatrix-assisted laser desorption ionization-time of flight massspectrometry (MALDI-TOF) (Wirth, U. et al. (2002) Proteomics 2(10):1445-51). The collection of proteins including a specific protein andall modified forms of it is referred to herein as a “protein cluster.”The collection of all modified forms of a specific protein, excludingthe specific protein, itself, is referred to herein as a “modifiedprotein cluster.” Modified forms of any cryptochrome also may be used,themselves, in the methods disclosed herein. In certain cases themodified forms may exhibit better discriminatory power in diagnosis thanthe specific forms set forth herein. Modified forms can be initiallydetected by any methodology known in the art.

Alternatively, cryptochrome protein and nucleic acid metabolites can bemeasured. The term “metabolite” includes any chemical or biochemicalproduct of a metabolic process, such as any compound produced by theprocessing, cleavage or consumption of a biological molecule (e.g., aprotein, nucleic acid, carbohydrate, or lipid). Metabolites can bedetected in a variety of ways known to one of skill in the art,including the refractive index spectroscopy (RI), ultra-violetspectroscopy (UV), fluorescence analysis, radiochemical analysis,near-infrared spectroscopy (near-IR), nuclear magnetic resonancespectroscopy (NMR), light scattering analysis (LS), mass spectrometry,pyrolysis mass spectrometry, nephelometry, dispersive Ramanspectroscopy, gas chromatography combined with mass spectrometry, liquidchromatography (including high-performance liquid chromatography(HPLC)), which may be combined with mass spectrometry, matrix-assistedlaser desorption ionization-time of flight (MALDI-TOF) combined withmass spectrometry, ion spray spectroscopy combined with massspectrometry, capillary electrophoresis, ion mobility spectrometry,surface-enhanced laser desorption/ionization (SELDI), optical methods,electrochemical methods, atomic force microscopy, radiofrequencymethods, surface Plasmon resonance, ellipsometry, NMR and IR detection.(See, International Application Publication Nos. WO 04/056456 and WO04/088309, each of which are hereby incorporated by reference in theirentireties). In this regard, other analytes can be measured using theabove-mentioned detection methods, or other methods known to the skilledartisan. For example, circulating calcium ions (Ca2+) can be detected ina sample using fluorescent dyes such as the Fluo series, Fura-2A,Rhod-2, among others. Other metabolites can be similarly detected usingreagents that specifically designed or tailored to detect suchmetabolites.

A Cry-mediated disease or disorder may involve changes in the activityof one or more cryptochromes, or ability of one or more cryptochromes tobind to a target. Without wishing to be bound by theory, cryptochromeproteins are believed to bind to Period proteins Per1 and/or Per2 as aheterodimer, which then bind to the promoter region of the CLOCK-BMAL1gene to facilitate transcriptional repression in a feedback loop thatcan impinge upon numerous metabolic processes. Thus, measuring aneffective amount of one or more cryptochromes according to the methodsof the invention may involve assessing an increase or decrease in theability of Cry proteins to bind to Per1 and/or Per2, to theglucocorticoid receptor (GR), or any other binding target of Cry knownto those skilled in the art. Measurement of protein-protein interactionsmay be facilitated by any method known in the art, includingco-immunoprecipitation, yeast two-hybrid assay, surface Plasmonresonance, bimolecular fluorescence complementation, tandem affinitypurification, phage display, fluorescence polarization/anisotropy, dualpolarization interferometry, fluorescence correlation spectroscopy,fluorescence resonance energy transfer, and the like.

The activity of one or more cryptochromes may also be measured by anincrease or decrease in the ability to bind to a DNA sequence, i.e., thepromoter region of the CLOCK-BMAL1 gene, or other gene that containsbinding sites recognized by one or more cryptochromes. “Promoter”,“promoter sequence”, or “promoter region” refers to a DNA sequencecapable of binding RNA polymerase in a cell, initiating transcription ofa downstream (3′ direction) coding sequence, thereby controlling itsexpression. For purposes of defining the present invention, the promotersequence is bounded at its 3′ terminus by the transcription initiationsite and extends upstream (5′ direction) to include the minimum numberof bases or elements necessary to initiate transcription at levelsdetectable above background. Within the promoter sequence will be founda transcription initiation site (conveniently defined for example, bymapping with nuclease S1), as well as protein binding domains (consensussequences) responsible for the binding of RNA polymerase. Promoters maybe derived in their entirety from a native gene, or be composed ofdifferent elements derived from different promoters found in nature, oreven comprise synthetic DNA segments. In most cases the exact boundariesof regulatory sequences have not been completely defined, DNA fragmentsof different lengths may have identical promoter activity.

The CLOCK-BMAL1 promoter (or any other promoter region containingbinding or recognition sites for Cry) may be “operably linked” to areporter gene. The term “operably linked” refers to the association ofnucleic acid sequences on a single nucleic acid fragment so that thefunction of one is affected by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of affectingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in sense or antisenseorientation. The term “reporter gene” means a nucleic acid encoding anidentifying factor that is able to be identified based upon the reportergene's effect, wherein the effect is used to track the inheritance of anucleic acid of interest, to identify a cell or organism that hasinherited the nucleic acid of interest, and/or to measure geneexpression induction or transcription. Examples of reporter genes knownand used in the art include: luciferase (Luc), green fluorescent protein(GFP), alkaline phosphatase (ALP), chloramphenicol acetyltransferase(CAT), β-galactosidase (LacZ), β-glucuronidase (Gus), and the like.Selectable marker genes may also be considered reporter genes. Thepromoter-reporter gene construct may be contained in a plasmid orexpression vector that is transferred or transfected into a cell. Theexpression of the reporter gene can be detected by determining theactivity of the gene product, for example, an enzyme activity in thecase of using a reporter gene exemplified above.

The term “plasmid” refers to an extra chromosomal element often carryinga gene that is not part of the central metabolism of the cell, andusually in the form of circular double-stranded DNA molecules. Suchelements may be autonomously replicating sequences, genome integratingsequences, phage or nucleotide sequences, linear, circular, orsupercoiled, of a single- or double-stranded DNA or RNA, derived fromany source, in which a number of nucleotide sequences have been joinedor recombined into a unique construction which is capable of introducinga promoter fragment and DNA sequence for a selected gene product alongwith appropriate 3′ untranslated sequence into a cell. The term“expression vector” means a vector, plasmid or vehicle designed toenable the expression of an inserted nucleic acid sequence followingtransformation into the host. Vectors may be introduced into the desiredhost cells by methods known in the art, e.g., transfection,electroporation, microinjection, transduction, cell fusion, DEAEdextran, calcium phosphate precipitation, lipofection (lysosome fusion),use of a gene gun, or a DNA vector transporter. Any cell may be used tocarry out reporter assays, such as a prokaryotic cell or eukaryoticcell. Preferably, the cell may be a bacterial cell, a fungal cell, ayeast cell, a nematode cell, an insect cell, a fish cell, a plant cell,an avian cell, an animal cell, and a mammalian cell. Cells may beprimary cells or may be continuously passaged as cell lines. Exemplarycells and cell lines are known to those skilled in the art.

Other methods of measuring the activity or ability of one or morecryptochromes to bind to a DNA sequence include chromatinimmunoprecipitation assay, electrophoretic mobility shift assay, DNApull-down assay, microplate capture and detection, and the like.

Levels of an effective amount of cryptochrome proteins, nucleic acids,polymorphisms, metabolites, or other analytes, or the activities ofcryptochrome proteins or targets that are directly or indirectly boundto cryptochrome proteins, can then be determined and compared to areference value, e.g. a control subject or population whose diseasestatus is known, or an index value or baseline value. The referencesample or index value or baseline value may be taken or derived from oneor more subjects who have been exposed to the treatment, or may be takenor derived from one or more subjects who are at low risk of developing aCry-mediated disease or disorder, or may be taken or derived fromsubjects who have shown improvements in disease risk factors as a resultof exposure to treatment. Alternatively, the reference sample or indexvalue or baseline value may be taken or derived from one or moresubjects who have not been exposed to the treatment. For example,samples may be collected from subjects who have received initialtreatment for a Cry-mediated disease or disorder and subsequenttreatment for the disease or disorder to monitor the progress of thetreatment. In some embodiments, a first sample may be taken from asubject at a first period of time, e.g., prior to treatment with acompound of formula I as defined herein, either alone or in combinationwith one or more additional therapeutic agents, followed by measuring ordetecting one or more cryptochromes (or cryptochrome targets) asdescribed herein. Thereafter, a second sample may be taken from asubject at a second period of time, e.g., after treatment with acompound of formula I as defined herein, either alone or in combinationwith one or more additional therapeutic agents, and measuring the one ormore cryptochromes or cryptochrome targets. Any number of samples may betaken at any time interval throughout the course of treatment to assessits effectiveness.

A reference value can also comprise a value derived from risk predictionalgorithms or computed indices from population studies such as thosedisclosed herein. A similar term in this context is a “control”, whichcan be, e.g., the average or median amount of cryptochromes present incomparable samples of normal subjects in normal subjects or innon-disease subjects such as where a Cry-mediated disease or disorder isundetectable. The control amount is measured under the same orsubstantially similar experimental conditions as in measuring the testamount. The correlation may take into account the presence or absence ofthe cryptochromes in a test sample and the frequency of detection of thesame molecules in a control. The correlation may take into account bothof such factors to facilitate determination of disease status.

A reference profile of those subjects who do not have a Cry-mediateddisease or disorder, and would not be expected to develop a Cry-mediateddisease or disorder may also be prepared according to methods disclosedherein. Measurement of one or more cryptochromes can also be used togenerate a “subject profile” taken from subjects who have a Cry-mediateddisease or disorder. The subject profiles can be compared to a referenceprofile to diagnose or identify subjects at risk for developing aCry-mediated disease or disorder, to monitor the progression of disease,as well as the rate of progression of disease, and to monitor theeffectiveness of treatment modalities or subject management.

The reference and subject profiles of the present invention can becontained in a machine-readable medium, such as but not limited to,analog or digital tapes like those readable by a VCR, CD-ROM, DVD-ROM,USB flash media, among others. Such machine-readable media can alsocontain additional test results, such as, without limitation,measurements of clinical parameters and traditional laboratory riskfactors. Alternatively or additionally, the machine-readable media canalso comprise subject information such as medical history and anyrelevant family history. The machine-readable media can also containinformation relating to other risk algorithms and computed indices suchas those described herein.

In any of the methods disclosed herein, the data from the sample may befed directly from the detection means into a computer containing thediagnostic algorithm. Alternatively, the data obtained can be fedmanually, or via an automated means, into a separate computer thatcontains the diagnostic algorithm. Accordingly, embodiments of theinvention include methods involving correlating the detection of thecryptochromes with a probable diagnosis of a Cry-mediated disease ordisorder. The correlation may take into account the amount of the one ormore cryptochromes in the sample compared to a control amount (up ordown regulation of the cryptochromes) (e.g., in normal subjects in whoma Cry-mediated disease or disorder is undetectable). The correlation maytake into account the presence or absence of the cryptochromes in a testsample and the frequency of detection of the same molecules in acontrol. The correlation may take into account both of such factors tofacilitate determination of whether a subject has a Cry-mediated diseaseor disorder or not.

Data analysis can include the steps of determining signal strength(e.g., height of peaks) of a marker detected and removing “outliers”(data deviating from a predetermined statistical distribution). Theobserved peaks can be normalized, a process whereby the height of eachpeak relative to some reference is calculated. For example, a referencecan be background noise generated by instrument and chemicals (e.g.,energy absorbing molecule) which is set as zero in the scale. The signalstrength detected for each molecule of interest can be displayed in theform of relative intensities in the scale desired (e.g., 100).Alternatively, a standard (e.g., a serum protein) may be admitted withthe sample so that a peak from the standard can be used as a referenceto calculate relative intensities of the signals observed for eachmolecule of interest detected.

The resulting data can be transformed or converted into various formatsfor displaying. In one format, referred to as “spectrum view orretentate map,” a standard spectral view can be displayed, wherein theview depicts the quantity of molecule reaching the detector at eachparticular molecular weight. In another format, referred to as “peakmap,” only the peak height and mass information are retained from thespectrum view, yielding a cleaner image and enabling molecules ofinterest with nearly identical molecular weights to be more easily seen.In yet another format, referred to as “gel view,” each mass from thepeak view can be converted into a grayscale image based on the height ofeach peak, resulting in an appearance similar to bands onelectrophoretic gels. In yet another format, referred to as “3-Doverlays,” several spectra can be overlaid to study subtle changes inrelative peak heights. In yet another format, referred to as “differencemap view,” two or more spectra can be compared, convenientlyhighlighting unique molecules of interest which are up- ordown-regulated between samples. Profiles (spectra) from any two samplesmay be compared visually. In yet another format, Spotfire Scatter Plotcan be used, wherein molecules of interest that are detected are plottedas a dot in a plot, wherein one axis of the plot represents the apparentmolecular weight of the cryptochromes detected and another axisrepresents the signal intensity of cryptochromes detected. For eachsample, molecules of interest that are detected and the amount ofmolecules present in the sample can be saved in a computer readablemedium. This data can then be compared to a control or reference profileor reference value (e.g., a profile or quantity of molecules detected incontrol, e.g., subjects in whom a Cry-mediated disease or disorder isundetectable).

The data that are generated in the methods disclosed herein can beclassified using a pattern recognition process that uses aclassification model. In some embodiments, data generated using samplessuch as “known samples” can then be used to “train” a classificationmodel. A “known sample” is a sample that is pre-classified (e.g.,disease or no disease). Data generated using known samples can then beused to “train” a classification model. A “known sample” is a samplethat is pre-classified. The data can be used to form the classificationmodel can be referred to as a “training data set”. Once trained, theclassification model can recognize patterns in data generated usingunknown samples. The classification model can then be used to classifythe unknown samples into classes. This can be useful, for example, inpredicting whether or not a particular biological sample is associatedwith a certain biological condition (e.g., diseased vs. non diseased).The training data set that is used to form the classification model maycomprise raw data or pre-processed data. In some embodiments, raw datacan be obtained directly from time-of-flight spectra or mass spectra,and then may be optionally “pre-processed” in any suitable manner.Pre-processing steps such as these can be used to reduce the amount ofdata that is used to train the classification model.

Classification models can be formed using any suitable statisticalclassification (or “learning”) method that attempts to segregate bodiesof data into classes based on objective parameters present in the data.Classification methods may be either supervised or unsupervised.Examples of supervised and unsupervised classification processes aredescribed in Jain, “Statistical Pattern Recognition: A Review”, IEEETransactions on Pattern Analysis and Machine Intelligence, Vol. 22, No.1, January 2000, which is herein incorporated by reference in itsentirety. In supervised classification, training data containingexamples of known categories are presented to a learning mechanism,which learns one more sets of relationships that define each of theknown classes. New data may then be applied to the learning mechanism,which then classifies the new data using the learned relationships.

Examples of supervised classification processes include linearregression processes (e.g., multiple linear regression (MLR), partialleast squares (PLS) regression and principal components regression(PCR)), binary decision trees (e.g., recursive partitioning processessuch as CART—classification and regression trees), artificial neuralnetworks such as back propagation networks, discriminant analyses (e.g.,Bayesian classifier or Fischer analysis), logistic classifiers, andsupport vector classifiers (support vector machines). A preferredsupervised classification method is a recursive partitioning process(U.S. Patent Application Publication No. 20020138208). Unsupervisedclassification attempts to learn classifications based on similaritiesin the training data set, without pre classifying the spectra from whichthe training data set was derived. Unsupervised learning methods includecluster analyses. A cluster analysis attempts to divide the data into“clusters” or groups that ideally should have members that are verysimilar to each other, and very dissimilar to members of other clusters.Similarity is then measured using some distance metric, which measuresthe distance between data items, and clusters together data items thatare closer to each other. Clustering techniques include the MacQueen'sK-means algorithm and the Kohonen's Self-Organizing Map algorithm.Learning algorithms asserted for use in classifying biologicalinformation are described in, for example, International ApplicationPublication No. WO 01/31580 and U.S. Patent Application Publication Nos.20020193950, 20030004402, and 20030055615. Another classification methodinvolves multivariate predictive models using a non-linear version ofUnified Maximum Separability Analysis (“USMA”) classifiers. Details ofUSMA classifiers are described in U.S. Patent Application PublicationNo. 20030055615.

Other classification algorithms and formulae include, but are notlimited to, Principal Component Analysis (PCA), cross-correlation,factor rotation, Logistic Regression (LogReg), Linear DiscriminantAnalysis (LDA), Eigengene Linear Discriminant Analysis (ELDA), RandomForest (RF), Recursive Partitioning Tree (RPART), as well as otherrelated decision tree classification techniques, Shrunken Centroids(SC), StepAIC, Kth-Nearest Neighbor, Boosting, Decision Trees, NeuralNetworks, Bayesian Networks, Support Vector Machines, Leave-One-Out(LOO), 10-Fold cross-validation (10-Fold CV), and Hidden Markov Models,among others.

Detection and correlation of one or more cryptochromes may also beanalyzed using any suitable means, including software packages, forexample, Applied Maths, GenExplore™, 2-way cluster analysis, principalcomponent analysis, discriminant analysis, self-organizing maps;BioDiscovery, Inc., Los Angeles, Calif. (ImaGene™, special imageprocessing and data extraction software, powered by MatLab®; GeneSight:hierarchical clustering, artificial neural network (SOM), principalcomponent analysis, time series; AutoGene™; CloneTracker™); GeneData AG(Basel, Switzerland); Molecular Pattern Recognition web site at MIT'sWhitehead Genome Center; Rosetta Inpharmatics, Kirkland, Wash. Resolver™Expression Data Analysis System; Scanalytics, Inc., Fairfax, Va. ItsMicroArray Suite enables researchers to acquire, visualize, process, andanalyze gene expression microarray data; TIGR (The Institute for GenomeResearch) offers software tools for array analysis. For example, seealso Eisen and Brown, (1999) Methods Enzymol. 303: 179-205.

In certain embodiments of the methods of qualifying disease status, themethods further comprise managing or modifying clinical treatment of asubject based on the status of the disease or disorder. For example, ifthe result of the methods of the present invention is inconclusive orthere is reason that confirmation of status is necessary, the physicianmay order more tests (e.g., CT scans, PET scans, MRI scans, PET-CTscans, X-rays, biopsies, blood tests. Alternatively, if the statusindicates that treatment is appropriate, the physician may schedule thesubject for treatment. In other instances, the subject may receivetherapeutic treatments (such as administration of therapeutic agents(such as, e.g., the compounds of formula I defined herein, either aloneor in combination with one or more additional therapeutic agents),either in lieu of, or in addition to, surgery. No further action may bewarranted. Furthermore, if the results show that treatment has beensuccessful, a maintenance therapy or no further management may benecessary.

The subject matter disclosed herein also provides for such methods wherethe cryptochromes are measured again after clinical treatment of asubject. In these cases, the methods are used to monitor the status of aCry-mediated disease or disorder, e.g., response to treatment, remissionof the disease or progression of the disease. The methods can berepeated after each treatment the subject receives, allowing thephysician to follow the effectiveness of the course of treatment. If theresults show that the treatment is not effective, the course oftreatment can be altered accordingly.

The invention provides kits for qualifying disease status and/ordetecting or diagnosing disease, wherein the kits can be used to detectone or more cryptochromes. For example, the kits can be used to detectany one or more of the cryptochromes described herein, which the one ormore cryptochromes are differentially present in samples of diseasesubjects and normal subjects. The kits of the invention have manyapplications. For example, the kits can be used in any one of themethods of the invention described herein, such as, inter alia, todifferentiate if a subject has a Cry-mediated disease or disorder or hasa negative diagnosis, thus aiding a diagnosis. In another example, thekits can be used to identify compounds that modulate expression of oneor more of the cryptochromes, compounds that modulate the activity ofone or more cryptochromes (i.e., that affect the ability of one or morecryptochromes to bind to a target such as Per1, Per2, the glucocorticoidreceptor (GR), or a promoter sequence recognized by cryptochromes suchas the CLOCK-BMAL1 promoter or any other promoter sequence) by using invitro or in vivo animal models for a Cry-mediated disease or disorder.In another example, the kits can be used to identify binding targets ofone or more cryptochrome proteins as defined herein.

Kits of the present invention may include a detection reagent, e.g.,nucleic acids that specifically identify one or more cryptochromenucleic acids by having homologous nucleic acid sequences, such asoligonucleotide sequences, primers, or aptamers, complementary to aportion of the nucleic acids or antibodies to proteins encoded by thenucleic acids packaged together. The oligonucleotides can be fragmentsof the genes. The oligonucleotides may be single stranded or doublestranded. For example the oligonucleotides can be 200, 150, 100, 50, 25,10 or less nucleotides in length. Alternatively, the detection reagentmay be one or more antibodies that specifically or selectively bind toone or more cryptochrome proteins or targets thereof. The kit maycontain in separate containers a nucleic acid or antibody (eitheralready bound to a solid matrix or packaged separately with reagents forbinding them to the matrix), control formulations (positive and/ornegative), and/or a detectable label such as fluorescein, greenfluorescent protein, rhodamine, cyanine dyes, Alexa dyes, luciferase,radiolabels, among others. Instructions (e.g., written, tape, VCR,CD-ROM, etc.) for carrying out the assay and for correlation to diseasestatus may be included in the kit.

For example, detection reagents can be immobilized on a solid matrixsuch as a porous strip to form at least one detection site. Themeasurement or detection region of the porous strip may include aplurality of sites containing a nucleic acid. A test strip may alsocontain sites for negative and/or positive controls. Alternatively,control sites can be located on a separate strip from the test strip.Optionally, the different detection sites may contain different amountsof immobilized nucleic acids, e.g., a higher amount in the firstdetection site and lesser amounts in subsequent sites. Upon the additionof test sample, the number of sites displaying a detectable signalprovides a quantitative indication of the amount of cryptochromespresent in the sample. The detection sites may be configured in anysuitably detectable shape and are typically in the shape of a bar or dotspanning the width of a test strip. The substrate array can be on, e.g.,a solid substrate, e.g., a “chip” as described in U.S. Pat. No.5,744,305. Alternatively, the substrate array can be a solution array,e.g., xMAP (Luminex, Austin, Tex.), Cyvera (Illumina, San Diego,Calif.), CellCard (Vitra Bioscience, Mountain View, Calif.) and QuantumDots' Mosaic (Invitrogen, Carlsbad, Calif.). The kit may also containreagents, and/or enzymes for amplifying or isolating sample DNA. Thekits may include reagents for real-time PCR, for example, TaqMan probesand/or primers, and enzymes.

In some embodiments, a kit comprises: (a) a substrate comprising anadsorbent thereon, wherein the adsorbent retains or is otherwisesuitable for binding a cryptochrome, and (b) instructions to detect thecryptochrome by contacting a sample with the adsorbent and detecting thecryptochrome retained by the adsorbent. In some embodiments, the kit maycomprise an eluant (as an alternative or in combination withinstructions) or instructions for making an eluant, wherein thecombination of the adsorbent and the eluant allows detection of thecryptochrome using gas phase ion spectrometry.

In other embodiments, the kit may comprise a first substrate comprisingan adsorbent thereon (e.g., a particle functionalized with an adsorbent)and a second substrate onto which the first substrate can be positionedto form a probe, which may be removed and inserted into machine, suchas, e.g., a gas phase ion spectrometer. In other embodiments, the kitmay comprise a single substrate, which is in the form of a probe withadsorbents on the substrate that can be removed and inserted into amachine. In yet another embodiment, the kit may further comprise apre-fractionation spin column (e.g., Cibacron blue agarose column,anti-HSA agarose column, K-30 size exclusion column, Q-anion exchangespin column, single stranded DNA column, lectin column, etc.). Inanother embodiment, a kit comprises (a) an antibody that specificallybinds to one or more cryptochromes; and (b) a detection reagent. Anantibody may be, for example, an antibody directed against the geneproducts of a cryptochrome gene.

Optionally, the kit may further comprise a standard or controlinformation so that the test sample can be compared with the controlinformation standard to determine if the test amount of one or morecryptochromes detected in a sample is a diagnostic amount consistentwith a diagnosis of a Cry-mediated disease or disorder.

Although a few variations have been described in detail above, othermodifications or additions are possible. In particular, further featuresand/or variations may be provided in addition to those set forth herein.For example, the implementations described above may be directed tovarious combinations and subcombinations of the disclosed featuresand/or combinations and subcombinations of several further featuresdisclosed above. In addition, the logic flow described herein does notrequire the particular order shown, or sequential order, to achievedesirable results. Other embodiments may be within the scope of theclaims.

EXAMPLES Example 1 Reaction Schemes for Synthesis of Compounds

The following reaction schemes, Reaction Scheme I, II, III, IV, V, andVI depicts methods of synthesis for compounds of formula I. In thegeneral methods for preparation of the compounds of formula I, thevariable R₁, R₂, R₃, R₄, R₅, R₆, R₇, a, and b are as previously definedfor a compound of formula I unless otherwise stated. The ReactionSchemes herein described are intended to provide a general descriptionof the methodology employed in the preparation of many of the compoundsgiven. However, it will be evident from the detailed descriptions thatthe modes of preparation employed extend further than the generalprocedures described herein. In particular, it is noted that thecompounds prepared according to the Schemes may be modified further toprovide new compounds within the scope of this invention. The reagentsand intermediates used in the following compounds are eithercommercially available or can be prepared according to the standardliterature procedures by those skilled in the art of organic synthesis.

Reaction Scheme I, below, depicts the synthesis of compounds of formulaI. Treatment of an appropriately substituted bromide derivative offormula IV with an appropriate carbazole of formula V, in an appropriatesolvent, such as N,N-dimethylformamide or N,N-dimethylacetamide, withina temperature range of approximately 0° C. to 150° C. for a period ofapproximately 5 minutes to 24 hours provides the corresponding oxiranecompound of formula III. Preferred conditions for reacting the bromidecompound of formula IV with the carbazole of formula V to providecompounds of formula III include carrying out the reaction inN,N-dimethylformamide at 0° C. to room temperature in the presence ofpotassium hydroxide for 20 to 24 hours followed by an extractive workup.Treatment of the compound of formula III with an appropriate amide orurea of formula II, in an appropriate solvent, such asN,N-dimethylformamide, dimethyl sulfoxide or N,N-dimethylacetamide,within a temperature range of approximately room temperature to 150° C.for a period of approximately 5 mins to 3 days provides thecorresponding amide or urea compound of formula I. Preferred conditionsfor reacting the oxirane compound of formula III to provide compounds offormula I include carrying out the reaction in N,N-dimethylformamidewith sodium hydride at room temperature for 20 to 24 hours followed byextractive workup. Alternatively, the oxirane compound of formula IIIcan be reacted with the amide or urea of formula II in an appropriatesolvent, such as dimethyl sulfoxide, with an appropriate base, such aspotassium tert-butoxide, at room temperature for 3 days to provide thecompound of formula I.

Reaction Scheme II, below, depicts an alternative synthesis of compoundsof formula I. Treatment of an appropriately substituted oxiranederivative of formula III with an appropriate diamine of formula VII, inan appropriate solvent, such as ethanol, within a temperature range of0° C. to 150° C. for a period of approximately 5 minutes to 24 hoursprovides the corresponding diamine compound of formula VI. Preferredconditions for reacting oxirane compound of formula III with the diamineof formula VII to provide compounds of formula VI include carrying outthe reaction in ethanol at 40° C. for 20 to 24 hours. Treatment of thecompound of formula VI with an appropriate carbonylating agent, such as1,1′-carbonyldiimidazole, in an appropriate solvent such astetrahydrofuran, at room temperature for a period of 5 minutes to 24hours provides the corresponding compound of formula I.

Reaction Scheme III, below, depicts an alternative synthesis ofcompounds of formula I. Treatment of the amide or urea compound offormula II with an appropriately substituted bromide derivative offormula IX, in an appropriate solvent, such as tetrahydrofuran, within atemperature range of approximately 0° C. to 65° C. for a period ofapproximately 5 minutes to 24 hours provides the corresponding oxiranecompound of formula VIII. Preferred conditions for reacting the bromidecompound of formula IX with the amide or urea of formula II to providecompounds of formula VIII include carrying out the reaction intetrahydrofuran at 0° C. to room temperature in the presence of sodiumhydride for 20 to 24 hours followed by an extractive workup. Treatmentof the compound of formula VIII with an appropriate carbazole of formulaV, in an appropriate solvent, such as N,N-dimethylformamide, within atemperature range of 0° C. to 70° C. for a period of approximately 5minutes to 24 hours provides the corresponding compound of formula I.Preferred conditions for reacting oxirane compound of formula VIII withthe carbazole of formula V to provide compounds of formula I includecarrying out the reaction in N,N-dimethylformamide at room temperatureto 70° C. in the presence of sodium hydride for 20 to 24 hours toprovide the compound of formula I.

Reaction Scheme IV, below, depicts an alternative synthesis of compoundsof formula I. Treatment of the Boc-protected amino acid compound offormula XIII with ammonia, an appropriate coupling reagent, such asN,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uroniumhexafluorophosphate, an appropriate base, such asN,N-diisopropylethylamine and an appropriate solvent, such asdimethylformamide, within a temperature range of approximately 0° C. to65° C. for a period of approximately 5 minutes to 24 hours provides thecorresponding amide compound of formula XII. Treatment of Boc-protectedamino amide compound of formula XII with an appropriate reducing agent,such as borane, in an appropriate solvent, such as tetrahydrofuran,within a temperature range of approximately 0° C. to 100° C. for aperiod of approximately 5 minutes to 24 hours provides the correspondingBoc-protected diamine compound of formula XI. Preferred conditions forreacting oxirane compound of formula III with the Boc-protected diamineof formula XI to provide compounds of formula X include carrying out thereaction in ethanol at 70° C. for 16 to 24 hours. Treatment of thecompound of formula X with an appropriate base, such as potassiumtert-butoxide, in an appropriate solvent, such as tetrayhdrofuran,within a temperature range of approximately 0° C. to 100° C. for aperiod of approximately 5 minutes to 24 hours provides the correspondingcompound of formula I.

Reaction Scheme V, below, depicts an alternative synthesis of compoundsof formula I. Treatment of the benzyl-protected diamine compound offormula XVI with an appropriate carbonylating agent, such as1,1′-carbonyldiimidazole, in an appropriate solvent, such astetrahydrofuran, at room temperature for a period of 5 minutes to 24hours provides the corresponding compound of formula XV. Preferredconditions for reacting oxirane compound of formula III with thebenzyl-protected urea of formula XV to provide compounds of formula XIVinclude carrying out the reaction in N,N-dimethylformamide at roomtemperature to 70° C. in the presence of sodium hydride for 16 to 24hours. Treatment of the benzyl-protected urea compound of formula XIVwith 1 to 50 psi hydrogen in the presence of an appropriate catalyst,such as palladium hydroxide on carbon, with an appropriate acid, such asacetic acid, in an appropriate solvent, such as tetrahydrofuran, withina temperature range of approximately room temperature to 100° C. for aperiod of approximately 5 minutes to 5 days provides the correspondingcompound of formula I.

Reaction Scheme VI, below, depicts an alternative synthesis of compoundsof formula I. Treatment of an appropriately substituted chiral oxiranederivative of formula XVII or XVIII with an appropriate amide or ureacompound of formula II, with an appropriate base, such as sodiumhydride, in an appropriate solvent, such as N,N-dimethylformamide ortetrahydrofuran, within a temperature range of 0° C. to 150° C. for aperiod of approximately 5 minutes to 24 hours provides the correspondingchiral amide or urea compound of formula I.

Reaction Scheme VII, below, depicts an alternative synthesis ofcompounds of formula I. Treatment of an appropriately substituted chiraloxirane derivative of formula XVII or XVIII with an appropriate diamineof formula VII, in an appropriate solvent, such as ethanol, within atemperature range of 0° C. to 150° C. for a period of approximately 5minutes to 24 hours provides the corresponding chiral diamine compoundof formula VI. Preferred conditions for reacting oxirane compound offormula XVII or XVIII with the diamine of formula VII to providecompounds of formula VI include carrying out the reaction in ethanol at55° C. for 5 to 24 hours. Treatment of the compound of formula VI withan appropriate carbonylating agent, such as 1,1′-carbonyldiimidazole, inan appropriate solvent such as tetrahydrofuran, at room temperature fora period of 5 minutes to 24 hours provides the corresponding compound offormula I.

In the reaction schemes described herein it is to be understood thathydroxyl groups in intermediates useful for preparing compounds offormula I may be protected by conventional groups known to those skilledin the art, as required. For example, intermediates containing ahydroxyl group may be protected as the correspondingtert-butyldimethylsilyl ether and subsequently deprotected by treatmentwith tetra-n-butylammonium fluoride to provide the free hydroxylderivative. Suitable protecting groups and methods for their removal areillustrated in “Protective Groups in Organic Synthesis”, 3^(rd) Ed., T.W. Greene and P. G. M. Wuts (Wiley & Sons, 1999).

¹H Nuclear magnetic resonance (NMR) spectra were in all cases consistentwith the proposed structures. Characteristic chemical shifts (δ) aregiven in parts-per-million downfield from tetramethylsilane usingconventional abbreviations for designation of major peaks: e.g. s,singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad.The mass spectra (m/z) were recorded using either electrosprayionization (ESI) or atmospheric pressure chemical ionization (APCI).Where thin layer chromatography (TLC) has been used it refers to silicagel TLC using silica gel 60 F₂₅₄ plates, R_(f) is the distance traveledby a compound divided by the distance traveled by the solvent front on aTLC plate. HPLC refers to high performance liquid chromatography.

The following specific examples are included for illustrative purposesand are not to be construed as a limitation to this disclosure.

Preparation of Intermediates Preparation 1:2-chloro-4-fluoro-N-(4-fluorophenyl)aniline

A round bottom flask was charged with 1-bromo-4-fluorobenzene (13.0 g,74.3 mmol), 2-chloro-4-fluoroaniline (11.354 g, 78.0 mmol), anhydroustoluene (200 mL) and potassium tert-butoxide (10.003 g, 89.1 mmol). Themixture was degassed and back-filled with nitrogen, and thentris(dibenzylideneacetone)dipalladium(0) (2.041 g, 2.2 mmol) andtri-tert-butylphosphine (0.902 g, 4.5 mmol) were added and the reactionwas stirred under nitrogen at 100° C. for 16 hours. After cooling, themixture was treated with 6 M aqueous hydrochloric acid to acidic pH andthen adjusted back to basic pH with solid sodium carbonate. The mixturewas dried (anhydrous magnesium sulfate), filtered through Celite and thefilter cake washed with ethyl acetate. The filtrate was concentrated andthe residue purified by silica gel chromatography (0-20% ethylacetate/hexanes) to afford a yellowish oil (14 g, 79%). ¹H NMR (300 MHz,CDCl₃): δ 7.14 (dd, 1H, J=8.4, 3.0 Hz), 7.12-6.98 (m, 5H), 6.88 (td, 1H,J=8.7, 3.0 Hz), 5.80 (br s, 1H).

Preparation 2: 3,6-difluoro-9H-carbazole

A mixture of potassium carbonate (26.528 g, 191.9 mmol),2-chloro-4-fluoro-N-(4-fluorophenyl)aniline (23.0 g, 96.0 mmol),tricyclohexylphosphonium tetrafluoroborate (3.534 g, 9.6 mmol),palladium diacetate (1.077 g, 4.8 mmol), and anhydrousN,N-dimethylacetamide (200 mL) was stirred under nitrogen at 130° C. for16 hours. After cooling, the mixture was concentrated and the residuetreated with ethyl acetate, filtered through Celite and the filter cakewashed with ethyl acetate. The filtrate was concentrated and the residuepurified by a short silica gel column (20-50% methylenechloride/hexanes) to afford the crude product which was recrystallizedfrom hexanes-methylene chloride to afford the pure product as a whitepowder (17.2 g, 88%). ¹H NMR (300 MHz, CDCl₃): δ 8.00 (br s, 1H), 7.67(dd, 2H, J=8.7, 2.7 Hz), 7.36 (dd, 2H, J=8.7, 4.2 Hz), 7.19 (td, 2H,J=9.0, 2.7 Hz).

Preparation 3: 9-(Oxiran-2-ylmethyl)-9H-carbazole

Powdered potassium hydroxide (3.36 g, 60 mmol) was added to a solutionof carbazole (8.36 g, 50 mmol) in anhydrous N,N-dimethylformamide (50mL) and stirred at ambient temperature for 1 hour. The reaction mixturewas cooled in an ice bath and epibromohydrin (10.3 mL, 125 mmol) wasadded. The ice bath was removed and the reaction was stirred at roomtemperature for 20 hours. The mixture was partitioned between ethylacetate and water. The organic layer was washed successively with waterand saturated aqueous sodium chloride solutions, dried (anhydrous sodiumsulfate), filtered, and concentrated. The crude material was trituratedwith hexanes, and recrystallized from ethyl acetate/hexanes to yield thedesired product as white needles (6.41 g, 58% yield). A second crop ofcrystals was crystallized from the mother liquor to give additionalproduct (1.2 g, 11%). ¹H NMR (300 MHz, CDCl₃) δ 8.11-8.08 (m, 2H),7.46-7.44 (m, 4H), 7.28-7.25 (m, 2H), 4.68-4.62 (dd, 1H, J=3.1, 15.8 Hz)4.45-4.38 (dd, 1H, J=4.8, 15.9 Hz), 3.37 (m, 1H), 2.84-2.81 (dd, 1H,J=4.2, 4.3 Hz), 2.60-2.57 (dd, 1H, J=2.5, 5.0 Hz); HPLC analysis: (C18,5-95% acetonitrile in water+0.1% trifluoroacetic acid over 20 min:retention time, % area at 254 nm): 7.83 min, 98.7%.

The following compounds were prepared analogously:

Structure Name Characterization

9-((2-methyloxiran-2- yl)methyl)-9H- carbazole ¹H NMR (300 MHz, CDCl₃):δ 8.10-8.07 (dt, 2H, J = 0.9, 7.5 Hz), 7.48-7.46 (m, 4H), 7.27-7.22 (m,2H), 4.63-4.58, 4.32-4.27 (ABq, 2H, J = 15.6 Hz), 2.69 (s, 2H), 1.34 (s,3H); HPLC analysis: (C18, 10-90% acetonitrile in water over 20 min:retention time, % area at 254 nm): 13.6 min, 97%.

3,6-difluoro-9- (oxiran-2-ylmethyl)- 9H-carbazole ¹H NMR (300 MHz,CDCl₃): δ 7.69 (dd, 2H, J = 8.7, 2.7 Hz), 7.39 (dd, 2H, J = 9.0, 3.9Hz), 7.24 (td, 2H, J = 9.0, 2.7 Hz), 4.68 (dd, 1H, J = 15.9, 3.0 Hz),4.32 (dd, 1H, J = 15.9, 5.1 Hz), 3.35 (m, 1H), 2.84 (t, 1H, J = 4.5 Hz),2.55 (dd, 1H, J = 4.5, 2.7 Hz); HPLC analysis: (C18, 10-90% acetonitrilein water + 0.1% trifluoroacetic acid over 20 min: retention time, % areaat 254 nm): 13.6 min, >98%.

3,6-difluoro-9-((2- methyloxiran-2- yl)methyl)-9H- carbazole ¹H NMR (300MHz, CDCl₃): δ 7.68 (dd, 2H, J = 8.4, 2.4 Hz), 7.41 (dd, 2H, J = 9.0,4.2 Hz), 7.23 (td, 2H, J = 9.0, 2.4 Hz), 4.62 and 4.22 (AB, 2H, J = 15.6Hz), 2.71 and 2.66 (AB, 2H, J = 4.5 Hz), 1.33 (s, 3H); HPLC analysis:(C18, 10-90% acetonitrile in water + 0.1% trifluoroacetic acid over 20min: retention time, % area at 254 nm): 14.2 min, 100%.

Preparation 4: 1-benzoylpyrrolidin-2-one

To a cold 0° C. solution of 2-pyrrolidinone (4.4 g, 51.7 mmol, 1.0equiv.) and triethylamine (15.4 mL, 111.2 mmol, 2.1 equiv.) in anhydroustetrahydrofuran (120 mL) was added 4-dimethylaminopyridine (0.075 g) andbenzoyl chloride (6.9 mL, 59.5 mmol, 1.1 equiv.). The resultant mixturewas stirred for 16 hrs at room temperature. The mixture was poured intowater and extracted with ethyl acetate. The organic fraction was washedwith 0.1 M aqueous hydrochloric acid, saturated aqueous sodiumbicarbonate, and saturated aqueous sodium chloride solutions, dried overanhydrous sodium sulfate, filtered and concentrated in vacuo to afford ared oil. The crude product was purified by silica gel columnchromatography, eluting with a gradient of 20-65% ethyl acetate inhexanes to give an off-white solid (5.63 g, 58%). ¹H NMR (300 MHz,CDCl₃): δ 7.61-7.57 (m, 2H), 7.53-7.47 (tt, 1H, J=1.5, 7.5 Hz),7.42-7.37 (m, 2H), 3.98-3.94 (t, 2H, J=7.1 Hz), 2.61-2.58 (t, 2H, J=8.0Hz), 2.20-2.10 (quint, 2H, J=7.5 Hz). ESI (m/z): 190.1 (M+H).

Preparation 5: 1-benzoyl-3-fluoropyrrolidin-2-one

To a −78° C. solution of 1-benzoylpyrrolidin-2-one (1 g, 5.3 mmol, 1.0equiv.) in anhydrous tetrahydrofuran (26 mL) was added lithiumdiisopropylamide (3.382 mL of a 2 M solution in tetrahydrofuran, 6.8mmol, 1.3 equiv.) and the mixture stirred at −78° C. for 30 mins. Asolution of N-fluorobenzene sulfonimide (2.5 g, 7.9 mmol, 1.5 equiv.) inanhydrous tetrahydrofuran (5 mL) was added slowly at −78° C. and thereaction stirred for 1 hr at −40° C. Saturated aqueous sodium hydrogencarbonate was added, the solution warmed to room temperature andextracted with ethyl acetate. The organic layer was washed withsaturated aqueous sodium chloride, dried over anhydrous sodium sulfate,filtered and concentrated in vacuo to afford a yellow solid. The crudeproduct was purified by silica gel column chromatography, eluting fromsilica gel with a gradient of 15-60% ethyl acetate in hexanes to give awhite solid (0.595 g, 54%). ¹H NMR (300 MHz, CDCl₃): δ 7.64-7.61 (m,2H), 7.57-7.52 (tt, 1H, J=1.5, 7.5 Hz), 7.45-7.39 (m, 2H), 5.28-5.06(dt, 1H, J=7.8, 51 Hz), 4.15-4.07 (m, 1H), 3.87-3.78 (m, 1H), 2.68-2.56(m, 1H), 2.45-2.27 (m, 1H). ¹⁹F NMR (282 MHz, CDCl₃): δ −188.9 to −189.2(ddd, J=12.1, 24.2, 51.8 Hz).

Preparation 6: 3-fluoropyrrolidin-2-one

To a solution of 1-benzoyl-3-fluoropyrrolidin-2-one (0.282 g, 1.4 mmol,1.0 equiv.) in anhydrous tetrahydrofuran (5 mL) was added octylamine(0.259 mL, 1.6 mmol, 1.1 equiv.) and the reaction was stirred for 16 hrsat room temperature. The reaction mixture was concentrated under reducedpressure to give a yellow oil. The crude product was purified by silicagel column chromatography eluting with a gradient of 70-100% ethylacetate in hexanes to afford a white solid. (0.104 g, 74% yield). ¹H NMR(300 MHz, CDCl₃): δ 7.81 (br s, 1H), 5.11-4.89 (ddd, 1H, J=6.3, 7.8,52.8 Hz), 3.49-3.42 (m, 1H), 3.36-3.27 (m, 1H), 2.57-2.41 (m, 1H),2.34-2.13 (m, 1H). ¹³C NMR (75 MHz, CDCl₃): δ 173.5-173.3 (d, J=20 Hz),89.9-87.4 (d, J=182 Hz), 39.1 (d, J=4 Hz), 28.6-28.4 (d, J=20 Hz). ¹⁹FNMR (282 MHz, CDCl₃): δ −190.1 to −190.4 (ddd, J=15, 27, 52 Hz).

Preparation 7: tert-butyl 2-oxopiperidine-1-carboxylate

To a stirred solution of piperidin-2-one (5 g, 50.4 mmol, 1.0 equiv.),triethylamine (14.022 mL, 100.9 mmol, 2.0 equiv.) andN,N-4-dimethylaminopyridine (0.123 g, 1.0 mmol) in methylene chloride(100 mL) at 0° C. was added di-tert-butyl dicarbonate (16.512 g, 75.7mmol, 1.5 equiv.). The mixture was slowly warmed to room temperature andstirred for 48 hrs. The reaction was quenched with water and the organiclayer was washed sequentially with 1 N aqueous hydrochloric acid,saturated aqueous sodium bicarbonate and saturated aqueous sodiumchloride, and dried over anhydrous sodium sulfate, filtered andconcentrated in vacuo. The residue was purified by silica gel column(0-100% ethyl acetate/hexanes) to afford the desired product as a yellowoil (8.5 g, 85%). ¹H NMR (300 MHz, CDCl₃): δ 3.72-3.62 (m, 2H),2.58-2.48 (m, 2H), 1.90-1.78 (m, 4H), 1.55 (s, 9H).

Preparation 8: tert-butyl 3-fluoro-2-oxopiperidine-1-carboxylate

To a stirred solution of tert-butyl 2-oxopiperidine-1-carboxylate (3 g,15.1 mmol, 1.0 equiv.) in anhydrous tetrahydrofuran (70 mL) undernitrogen at −78° C. was added sodium bis(trimethylsilyl)amide (22.586 mLof a 1 M solution in tetrahydrofuran, 22.6 mmol, 1.5 equiv.) dropwiseover a period of 30 mins. The resultant solution was stirred for 45 minsat −78° C., and then a solution of N-fluorobenzene sulfonimide (7.122 g,22.6 mmol, 1.5 equiv.) in anhydrous tetrahydrofuran (30 mL) was addeddropwise over a period of 30 mins. The reaction was stirred at −78° C.for 1 hr and then allowed to slowly warm to room temperature over 2 hrsand stirred at room temperature for 1 hr. The reaction was quenched withsaturated aqueous ammonium chloride and extracted with ethyl acetate.The organic phase was washed with saturated aqueous sodium chloride anddried over anhydrous magnesium sulfate, filtered and concentrated invacuo. The residue was treated with diethyl ether and the solids werediscarded. The solution was concentrated and the residue was purified bysilica gel column chromatography (0-100% ethyl acetate/hexanes) toafford the crude product fractions and the difluoro byproduct as a whitesolid (1.5 g). The crude product fraction was further purified by asecond run of silica gel chromatography to afford the desired product asa thick oil (0.46 g, 14%). ¹H NMR (300 MHz, CDCl₃): δ 4.92 (ddd, 1H,J=47.4, 8.7, 6.3 Hz), 3.78-3.60 (m, 2H), 2.35 (m, 1H), 2.15-1.80 (m,3H), 1.55 (s, 9H). ¹⁹F NMR (282 MHz, CDCl₃): δ −185.2 (dt, J=45.7, 15.5Hz).

Preparation 9: 3-fluoropiperidin-2-one

To a 0° C. solution of tert-butyl 3-fluoro-2-oxopiperidine-1-carboxylate(0.450 g, 2.1 mmol, 1.0 equiv.) in methylene chloride (5 mL) was addedtrifluoroacetic acid (1 mL, 13.5 mmol, 6.5 equiv.) and the resultantsolution was stirred for 3 hrs. The reaction was concentrated underreduced pressure and the residue was purified by silica gel column(0-100% ethyl acetate/hexanes and then 0-20% methanol/ethyl acetate) toafford the desired product as a white powder (0.23 g, 95%). ¹H NMR (300MHz, CDCl₃): δ 6.36 (br s, 1H), 4.85 (ddd, 1H, J=46.8, 8.1, 5.4 Hz),3.50-3.20 (m, 2H), 2.40-1.70 (m, 4H). ¹⁹F NMR (282 MHz, CDCl₃): δ −186.5(dt, J=46.5, 15.5 Hz).

Preparation 10: 3,3-difluoropiperidin-2-one

3,3-Difluoropiperidin-2-one was prepared according to the reportedprocedures (Kim, B. C. et al. Synthesis 2012, 44, 3165-3170).

Preparation 11: 1-benzoyl-3,3-difluoropyrrolidin-2-one

To a −78° C. solution of 1-benzoyl-3-fluoropyrrolidin-2-one fromPreparation 21B (0.3 g, 1.4 mmol, 1.0 equiv.) and N-fluorobenzenesulfonimide (0.639 g, 2.0 mmol, 1.4 equiv.) in anhydrous tetrahydrofuran(10 mL) was added lithium diisopropylamide (0.905 mL of a 2 M solutionin tetrahydrofuran, 1.8 mmol, 1.3 equiv.) and the mixture stirred at−78° C. for 30 mins. Additional portions of lithium diisopropylamidesolution (0.5 equiv.) and N-fluorobenzene sulfonimide (0.5 equiv. in 0.5mL of anhydrous tetrahydrofuran) were added and the mixture stirred for1 hr at −78° C. Saturated aqueous sodium hydrogen carbonate was added,the mixture warmed to room temperature and extracted with ethyl acetate.The organic layer was washed with saturated aqueous sodium chloride,dried over anhydrous sodium sulfate, filtered and concentrated in vacuo.The crude product was purified by silica gel column chromatography,eluting with a gradient of 15-50% ethyl acetate in hexanes to give awhite solid (0.09 g, 23%). ¹H NMR (300 MHz, CDCl₃): δ 7.66-7.61 (m, 2H),7.59-7.55 (m, 1H), 7.47-7.44 (m, 2H), 4.02-3.97 (m, 2H), 2.70-2.56 (tt,2H, J=6.6, 14.7 Hz). ¹⁹F NMR (282 MHz, CDCl₃): δ −106.0 to −106.1 (t,J=15 Hz).

Preparation 12: 3,3-difluoropyrrolidin-2-one

To a solution of 1-benzoyl-3,3-difluoropyrrolidin-2-one (0.085 g, 0.4mmol, 1.0 equiv.) in anhydrous tetrahydrofuran (1 mL) was addedoctylamine (0.075 mL, 0.5 mmol, 1.1 equiv.) and the reaction was stirredfor 16 hrs at room temperature. The mixture was concentrated underreduced pressure to afford a yellow oil. The crude residue was purifiedby silica gel column chromatography eluting with a gradient of 50-100%ethyl acetate in hexanes to give a white solid (0.024 g, 52% yield). ¹HNMR (300 MHz, CDCl₃): δ 7.93 (br s, 1H), 3.50-3.46 (br t, 2H, J=6.0 Hz),2.63-2.48 (tt, 2H, J=6.6, 15.2 Hz). ¹⁹F NMR (282 MHz, CDCl₃): δ −107.33to −107.44 (t, J=15.2 Hz). ¹³C NMR (75 MHz, CDCl₃): δ 167.5-166.7 (t,J=31 Hz), 121.1-114.4 (t, J=248 Hz), 37.1 (t, J=3.3 Hz), 31.2-30.6 (t,J=23.1 Hz).

Preparation 13: 1-benzyl-3-methylpyrrolidin-2-one; General procedure

To a cold −78° C. solution of 1-benzyl-2-pyrrolidinone (0.422 g, 2.4mmol, 1.0 equiv.) in anhydrous tetrahydrofuran (15 mL) was added lithiumdiisopropylamide (2.4 mL of a 2 M solution, 4.8 mmol, 2.0 equiv.) andthe resultant red solution was stirred for 30 mins at −78° C., andiodomethane (0.6 mL, 9.6 mmol, 4.0 equiv.) was added. The solution wasstirred at −78° C. for 1 hr and allowed to slowly warm to roomtemperature for 16 hrs. Saturated aqueous ammonium chloride was addedand the mixture extracted with ethyl acetate. The organic fraction waswashed with saturated aqueous sodium chloride, dried over anhydroussodium sulfate, filtered and concentrated in vacuo. The crude productwas purified by silica gel column chromatography, eluting with agradient of 35-80% ethyl acetate in hexanes to afford the product as atan liquid (0.374 g, 82%). ¹H NMR (300 MHz, CDCl₃): δ 7.37-7.34 (m, 5H),4.52-4.41 and 4.46-4.41 (ABq, 2H, J=14.6 Hz), 3.27-3.15 (m, 2H),2.60-2.46 (m, 1H), 2.28-2.15 (m, 1H), 1.68-1.58 (m, 1H), 1.28-1.25 (d,3H, J=7.2 Hz).

The following compounds were prepared analogously:

Structure Name Characterization

1-benzyl-3- isopropylpyrrolidin-2- one ¹H NMR (300 MHz, CDCl₃): δ7.34-7.20 (m, 5H), 4.56- 4.51 and 4.39-4.34 (ABq, 2H, J = 14.6 Hz),3.18-3.13 (m, 2H), 2.51-2.43 (td, 1H, J = 4.5, 9.0 Hz), 2.33-2.22 (m,1H), 2.05-1.92 (m, 1H), 1.83-1.74 (m, 1H), 1.02-1.00 (d, 3H, J = 6.6Hz), 0.89-0.87 (d, 3H, J = 6.6 Hz).

1-benzyl-3- cyclopentylpyrrolidin- 2-one ¹H NMR (300 MHz, CDCl₃): δ7.34-7.19 (m, 5H), 4.54- 4.49 and 4.39-4.34 (ABq, 2H, J = 14.6 Hz),3.20-3.13 (m, 2H), 2.57-2.49 (m, 1H), 2.26-1.90 (m, 4H), 1.77-1.51 (m,5H), 1.41-1.19 (m, 2H); ESI (m/z): 244.2 (M + H).

1-benzyl-3- methylpiperidin-2-one ¹H NMR (300 MHz, CDCl₃) δ 7.34-7.20(m, 5H), 4.68- 4.63, 4.52-4.47 (ABq, 2H, J = 14.7 Hz), 3.23-3.18 (dd,2H, J = 5.3, 7.2 Hz), 2.52-2.45 (m, 1H), 2.02-1.68 (m, 3H), 1.59-1.47(m, 1H), 1.31-1.29 (d, 3H, J = 7.2 Hz); ESI (m/z): 204.1 (M + H).

1-benzyl-3- isopropylpiperidin-2- one ¹H NMR (300 MHz, CDCl₃): δ7.33-7.21 (m, 5H), 4.71- 4.65 and 4.56-4.21 (ABq, 2H, J = 14.7 Hz),3.20-3.16 (m, 2H), 2.69-2.63 (m, 1H), 2.36-2.29 (m, 1H), 1.91-1.48 (m,4H), 0.99-0.97 (d, 3H, J = 6.9 Hz), 0.88-0.86 (d, 3H, J = 6.9 Hz); ESI(m/z): 232.2 (M + H).

1-benzyl-3- ethylpiperidin-2-one ¹H NMR (300 MHz, CDCl₃): δ 7.33-7.21(m, 5H), 4.58 (s, 2H), 3.21-3.17 (dd, 2H, J = 5.0, 6.9 Hz), 2.05-1.53(m, 6H), 1.00-0.95 (t, 3H, J = 7.5 Hz); ESI (m/z): 218.2 (M + H).

1-benzyl-3- cyclopentylpiperidin- 2-one ¹H NMR (300 MHz, CDCl₃): δ7.33-7.20 (m, 5H), 4.71- 4.67 and 4.51-4.46 (ABq, 2H, J = 14.7 Hz),3.20-3.16 (m, 2H), 2.52-2.39 (m, 2H), 1.94-1.54 (m, 10H), 1.53- 1.19 (m,2H); ESI (m/z): 258.2 (M + H).

1-benzyl-3-(cyclohex- 2-en-1-yl)piperidin-2- one ESI (m/z): 270.2 (M +H).

Preparation 14: 1-benzyl-3-cyclohexylpiperidin-2-one

Under a nitrogen atmosphere, 10% palladium on carbon (0.09 g) was addedto a solution of 1-benzyl-3-(cyclohex-2-en-1-yl)piperidin-2-one (0.6 g,2.3 mmol) in ethanol (10 mL). The mixture was placed under an atmosphereof hydrogen and stirred for 2 days. The suspension was filtered throughCelite and concentrated under reduced pressure to afford the desiredproduct as a clear liquid (0.578 g, 98%). ¹H NMR (300 MHz, CDCl₃): δ7.33-7.22 (m, 5H), 4.66-4.61 and 4.59-4.54 (ABq, 2H, J=14.7 Hz),3.19-3.14 (m, 2H), 2.34-2.21 (m, 2H), 1.86-1.52 (m, 9H), 1.39-1.04 (m,5H); ESI (m/z): 272.2 (M+H).

Preparation 15: 1-benzyl-3-phenylpiperidin-2-one

Synthesized according to the procedure from de Filippis, A. et al.Tetrahedron, 2004, 60, 9757. To a cold (−20° C.) stirred solution ofN-benzyl-2-piperidinone (1.326 g, 7.0 mmol, 2.2 equiv.) in anhydroustetrahydrofuran (14 mL, 0.5 M) was added lithiumbis(trimethylsilyl)amide (6.4 mL of a 1 M solution in anhydroustetrahydrofuran, 6.4 mmol, 2.0 equiv.) and the mixture was stirred for20 mins at −20° C. A solution of zinc chloride (0.955 g, 7.0 mmol, 2.2equiv.) in anhydrous tetrahydrofuran (8 mL) was added and the solutionstirred for 20 mins at −20° C. The resulting solution was cannulatedinto a solution of2-dicyclohexylphosphino-2′-(N,N-dimethylamino)biphenyl (0.094 g),tris(dibenzylideneacetone)dipalladium(0) (0.092 g), and bromobenzene(0.335 mL, 3.2 mmol, 1.0 equiv.) in anhydrous tetrahydrofuran (6 mL),and the resultant mixture was heated at 70° C. for 6 hrs. The reactionwas quenched with aqueous ammonium chloride and extracted with ethylacetate. The organic fraction was washed with saturated aqueous sodiumchloride, dried over anhydrous sodium sulfate, filtered and concentratedin vacuo. The crude product was purified by silica gel columnchromatography, eluting with a gradient of 15-60% ethyl acetate inhexanes to give a yellow liquid (0.629 g, 74%). ¹H NMR (300 MHz, CDCl₃):δ 7.38-7.21 (m, 10H), 4.74-4.69 and 4.66-4.61 (AB, 2H, J=14.4 Hz),3.77-3.72 (dd, 1H, J=6.0, 8.1 Hz), 3.41-3.28 (m, 2H), 2.23-2.13 (m, 1H),2.05-1.69 (m, 3H).

Preparation 16: 3-methylpyrrolidin-2-one

Trifluoromethanesulfonic acid (0.604 mL, 6.8 mmol, 4.0 equiv.) was addedto a solution of 1-benzyl-3-methylpyrrolidin-2-one (0.323 g, 1.7 mmol,1.0 equiv.) in toluene (2 mL, 1 M). The mixture was heated at 195° C. ina microwave reactor for 25 mins. The mixture was poured into a smallamount of saturated aqueous sodium bicarbonate, extracted with ethylacetate, washed with saturated aqueous sodium chloride, and the combinedaqueous layers extracted again with ethyl acetate. The combined organicfractions were dried over anhydrous sodium sulfate, filtered, andconcentrated in vacuo. The crude residue was purified by silica gelcolumn chromatography eluting with 0-10% methanol in methylene chlorideto give the desired product (0.087 g). ¹H NMR (300 MHz, CDCl₃): δ 6.49(br s), 3.37-3.26 (m, 2H), 2.53-2.28 (m, 2H), 1.80-1.65 (m, 1H),1.21-1.19 (d, 3H, J=6.6 Hz).

The following compounds were prepared analogously:

Structure Name Characterization

3-isopropylpyrrolidin-2- one ¹H NMR (300 MHz, CDCl₃): δ 6.01 (br s, 1H),3.30- 3.28 (m, 2H), 2.39-2.32 (m, 1H), 2.27-2.05 (m, 2H), 1.99-1.86 (m,1H), 1.02-0.99 (d, 3H, J = 6.6 Hz), 0.91- 0.88 (d, 3H, J = 6.6 Hz). ESI(m/z): 128.2 (M + H).

3-phenylpiperidin-2-one ¹H NMR (300 MHz, CDCl₃): δ 7.38-7.13 (m, 5H),6.02 (br s, 1H), 3.67-3.63 (dd, 1H, J = 6.3, 8.3 Hz), 3.49-3.41 (m, 2H),2.25-1.75 (m, 4H), ESI (m/z): 176.2 (M + H).

3-methylpiperidin-2-one ¹H NMR (300 MHz, CDCl₃): δ 5.83 (br s, 1H),3.33- 3.28 (m, 2H), 2.40-2.32 (m, 1H), 2.02-1.69 (m, 3H), 1.59-1.46 (m,1H), 1.26-1.24 (d, 3H, J = 7.2 Hz).

3-cyclopentylpyrrolidin- 2-one ¹H NMR (300 MHz, CDCl₃): δ 5.56 (br s,1H), 3.34- 3.28 (m, 2H), 2.44-2.36 (m, 1H), 2.27-2.13 (m, 2H), 1.96-1.87(m, 2H), 1.77-1.54 (m, 5H), 1.40-1.26 (m, 2H). ESI (m/z): 154.2 (M + H).

3-ethylpiperidin-2-one ¹H NMR (300 MHz, CDCl₃): δ 5.91 (br s, 1H), 3.31-3.26 (m, 2H), 2.27-2.17 (m, 1H), 1.99-1.47 (m, 6H), 1.00-0.93 (t, 3H, J= 7.8 Hz).

3-cyclopentylpiperidin-2- one ¹H NMR (300 MHz, CDCl₃): δ 5.75 (br s,1H), 3.31- 3.24 (m, 2H), 2.46-2.29 (m, 2H), 1.94-1.19 (m, 12H); ESI(m/z): 168.2 (M + H).

3-isopropylpiperidin-2- one ¹H NMR (300 MHz, CDCl₃): δ 5.92 (br s, 1H),3.34- 3.15 (m, 2H), 2.60-2.49 (m, 1H), 2.28-2.21 (m, 1H), 1.94-1.47 (m,4H), 0.98-0.95 (d, 3H, J = 6.9 Hz), 0.88- 0.85 (d, 3 Hz, J = 7.2 Hz);ESI (m/z): 142.2 (M + H).

3-cyclohexylpiperidin-2- one ¹H NMR (300 MHz, CDCl₃): δ 5.89 (br s, 1H),3.30- 3.19 (m, 2H), 2.26-2.10 (m, 2H), 1.91-1.03 (m, 14H); ESI (m/z):182.2 (M + H).

Preparation 17: 1-benzyl-3-(1-hydroxycyclobutyl)pyrrolidin-2-one

To a cold (−78° C.) solution of 1-benzyl-2-pyrrolidinone (1.0 g, 5.7mmol, 1.0 equiv.) in anhydrous tetrahydrofuran (19 mL) was added lithiumdiisopropylamide (3.15 mL of a 2 M solution in tetrahydrofuran, 1.1 eq.)and the mixture stirred for 1 hr at −78° C. Cyclobutanone (0.426 mL, 5.7mmol, 1.0 equiv.) and boron trifluoride diethyl etherate (0.704 mL, 5.7mmol, 1.0 equiv.) were added and the reaction mixture stirred at −78° C.for 4 hrs. The reaction was quenched with saturated aqueous ammoniumchloride and extracted with ethyl acetate. The organic fraction waswashed with saturated aqueous sodium chloride, dried over anhydroussodium sulfate, filtered and concentrated under reduced pressure. Thecrude product was purified by silica gel column chromatography elutingfrom silica gel with a gradient of 50-100% ethyl acetate in hexanes togive a white solid (0.714 g, 51%). ¹H NMR (300 MHz, CDCl₃): δ 7.36-7.20(m, 5H), 4.56-4.51 and 4.42-4.37 (AB, 2H, J=14.7 Hz), 4.24 (s, 1H),3.28-3.21 (m, 2H), 2.78-2.72 (t, 1H, J=2.7 Hz), 2.34-1.89 (m, 7H),1.66-1.52 (m, 1H); ESI (m/z): 246.0 (M+H).

The following compound was prepared analogously:

Structure Name Characterization

1-benzyl-3-(1- hydroxycyclobutyl)piperidin- 2-one ¹H NMR (300 MHz,CDCl₃): δ 7.38-7.21 (m, 5H), 5.35 (s, 1H), 4.67-4.62 and 4.55-4.50 (ABq,2H, J = 14.3 Hz), 3.26-3.22 (m, 2H), 2.55-2.49 (m, 2H), 2.37-2.34 (m,1H), 2.33-2.28 (m, 1H), 2.20-1.57 (m, 9H); ESI (m/z): 260.1 (M + H).

Preparation 18: 1-benzyl-3-cyclobutylidenepyrrolidin-2-one

To a cold (0° C.) solution of1-benzyl-3-(1-hydroxycyclobutyl)pyrrolidin-2-one (0.7 g, 2.9 mmol, 1.0equiv.) in anhydrous methylene chloride (12 mL) was addedN,N-diisopropylethylamine (2.485 mL, 14.3 mmol, 5.0 equiv.),N,N-4-dimethylaminopyridine (0.07 g, 0.6 mmol, 0.2 equiv.), andmethanesulfonyl chloride (0.331 mL, 4.3 mmol, 1.5 equiv.). The mixturewas stirred at 0° C. for 2 hrs, 16 hrs at room temperature, and atreflux for 3 hrs. The mixture was cooled to room temperature andpartitioned between ethyl acetate and saturated aqueous ammoniumchloride. The organic layer was washed with saturated aqueous sodiumchloride, dried over anhydrous sodium sulfate, filtered and concentratedin vacuo to afford an orange oil. The crude product was purified bysilica gel column chromatography, eluting with a gradient of 20-60%ethyl acetate in hexanes to give a yellow oil (0.25 g, 38%). ¹H NMR (300MHz, CDCl₃): δ 7.33-7.23 (m, 5H), 4.47 (s, 2H), 3.27-3.19 (m, 4H),2.75-2.69 (m, 2H), 2.52-2.46 (m, 2H), 2.18-2.08 (quint, 2H, J=7.8 Hz);ESI (m/z): 228.2 (M+H).

The following compound was prepared analogously:

Structure Name Characterization

1-benzyl-3- cyclobutylidenepiperidin-2- one ¹H NMR (300 MHz, CDCl₃): δ7.30-7.23 (m, 5H), 4.62 (s, 2H), 3.28-3.22 (m, 4H), 2.78-2.72 (m, 2H),2.34-2.29 (m, 2H), 2.12-2.02 (quint, 2H, J = 6.9 Hz), 1.82-1.76 (m, 2H);ESI (m/z): 242.2 (M + H).

Preparation 19: 1-benzyl-3-cyclobutylpyrrolidin-2-one

To a solution of 1-benzyl-3-cyclobutylidenepyrrolidin-2-one (0.25 g, 1.0mmol) in ethanol (11 mL) was added 10% palladium on carbon (0.05 g), andthe reaction mixture stirred under an atmosphere of hydrogen for 72 hrs.The mixture was filtered through Celite and concentrated under reducedpressure to afford a clear oil (0.242 g, 100%). ¹H NMR (300 MHz, CDCl₃):δ 7.34-7.20 (m, 5H), 4.49-4.47 and 4.40-4.35 (ABq, 2H, J=14.4 Hz),3.18-3.13 (m, 2H), 2.69-2.47 (m, 2H), 2.20-1.64 (m, 8H); ESI (m/z):230.2 (M+H).

The following compound was prepared analogously:

Structure Name Characterization

1-benzyl-3- cyclobutylpiperidin- 2-one (300 MHz, CDCl₃): δ 7.34-7.20 (m,5H), 4.55 (s, 2H), 3.19-3.14 (dd, 2H, J = 5.4, 6.9 Hz), 2.71-2.59 (m,1H), 2.39-1.47 (m, 11H).

Preparation 20: 3-cyclobutylpyrrolidin-2-one

Prepared from 1-benzyl-3-cyclobutylpyrrolidin-2-one analogously toPreparation 18. ¹H NMR (300 MHz, CDCl₃): δ 5.45 (br s, 1H), 3.34-3.27(m, 2H), 2.65-2.54 (m, 1H), 2.43-2.35 (m, 1H), 2.28-1.79 (m, 8H).

The following compound was prepared analogously:

Structure Name ¹H NMR

3-cyclobutylpiperidin-2-one (300 MHz, CDCl₃): δ 5.47 (br s, 1H),3.30-3.23 (m, 2H), 2.70-2.56 (m, 1H), 2.30-1.66 (m, 10H), 1.57-1.44 (m,1H).

Preparation 21: N1-cyclohexylethane-1,2-diamine

To a mixture of cyclohexanone (6.26 g, 63.8 mmol), ethylenediamine(42.64 mL, 637.8 mmol, 10.0 equiv.), acetic acid (36.515 mL, 637.8 mmol,10.0 equiv.), and 4 Å molecular sieves (25 g) in anhydrous methanol (250mL) was added sodium cyanoborohydride (8.017 g, 127.6 mmol, 2.0 equiv.).The mixture was stirred for 48 hrs, filtered to remove solids, andconcentrated to a semi-solid. The crude material was dissolved in 3 Naqueous sodium hydroxide (150 mL) and extracted with methylene chloridethree times. The combined organic fractions were washed with a slightlybasic saturated aqueous sodium chloride solution, dried over anhydroussodium sulfate, filtered, and concentrated in vacuo to afford a paleyellow liquid, which was purified by vacuum distillation to give a clearliquid (4.1 g, 45%). ¹H NMR (300 MHz, CDCl₃): δ 2.80-2.76 (td, 2H,J=0.9, 6.0 Hz), 2.68-2.64 (td, 2H, J=0.9, 6.0 Hz), 2.43-2.34 (m, 1H),1.89-1.83 (m, 2H), 1.74-1.70 (m, 2H), 1.62-1.57 (m, 1H), 1.32-0.98 (m,8H).

Preparation 22: 3-(cyclobutylamino)propanenitrile

At room temperature, cyclobutylamine (5.90 mL, 59.8 mmol, 1.0 equiv.)was added dropwise, over 15 mins to a solution of acrylonitrile (4.76 g,89.7 mmol, 1.5 equiv.) in methanol (7 mL). The mixture was stirred atroom temperature for 30 mins and at reflux for 1 hr, cooled to roomtemperature, concentrated under reduced pressure and the desired productdistilled under vacuum to provide a clear liquid (7.7 g, 98%). ¹H NMR(300 MHz, CDCl₃): δ 3.29-3.21 (m, 1H), 2.88-2.83 (t, 2H, J=6.6 Hz),2.50-2.46 (t, 2H, J=6.6 Hz), 2.26-2.20 (m, 2H), 1.76-1.63 (m, 4H), 1.30(br s, 1H).

Preparation 23: N1-cyclobutylpropane-1,3-diamine

To a cooled (0° C.) suspension of lithium aluminum hydride (3.056 g,80.5 mmol, 2.0 equiv.) in anhydrous ether (120 mL) was added a solutionof 3-(cyclobutylamino)propanenitrile (5.0 g, 40.3 mmol, 1.0 equiv.) inanhydrous diethyl ether (40 mL) dropwise over 45 mins. The reactionmixture was stirred at room temperature for 15 mins and at reflux for 4hrs, cooled to room temperature and stirred for 1 hr. The mixture wascooled to 0° C. and vigorously stirred while water (3.1 mL) was addeddropwise, followed by 15% aqueous sodium hydroxide (3.1 mL), and finallywater (9.3 mL). The resultant slurry was warmed to room temperature,stirred for 15 mins, and magnesium sulfate was added, stirring foradditional 15 mins. Solid materials were removed by filtration through aglass fritted filter, washing multiple times with warm methylenechloride, and the organic solution was concentrated under reducedpressure to give the desired product as a pale yellow liquid (3.44 g,66%). ¹H NMR (300 MHz, CDCl₃): δ 3.14 (m, 1H), 2.69-2.62 (m, 2H),2.53-2.45 (m, 2H), 2.13-2.10 (m, 2H), 1.56-1.48 (m, 6H), 1.33 (br s,3H).

Preparation 24: 3-(cyclopentylamino)propanenitrile

At room temperature, cyclopentylamine (5.794 mL, 58.7 mmol, 1.0 equiv.)was added dropwise to a solution of acrylonitrile (5.79 mL, 88.1 mmol,1.5 equiv.) in methanol (7 mL). The solution was stirred at roomtemperature for 30 mins and at reflux for 1 hr, cooled to roomtemperature, concentrated under reduced pressure and the desired productdistilled under vacuum to provide a clear liquid (7.4 g, 91%). ¹H NMR(300 MHz, CDCl₃): δ 3.14-3.04 (quin, 1H, J=6.3 Hz), 2.91-2.87 (t, 2H,J=6.9 Hz), 2.53-2.48 (td, 2H, J=0.9, 6.9 Hz), 1.88-1.78 (m, 2H),1.73-1.49 (m, 4H), 1.36-1.24 (m, 2H), 1.19 (br s, 1H).

Preparation 25: N1-cyclopentylpropane-1,3-diamine

To a cooled (0° C.) suspension of lithium aluminum hydride (3.295 g,86.8 mmol, 2.0 equiv.) in anhydrous diethyl ether (150 mL) was added asolution of 3-(cyclopentylamino)propanenitrile (6.0 g, 43.4 mmol, 1.0equiv.) in anhydrous diethyl ether (40 mL), dropwise over 45 mins. Thereaction mixture was stirred at room temperature for 15 mins and atreflux for 4 hrs, cooled to room temperature and stirred for 1 hr. Themixture was cooled to 0° C. and vigorously stirred while water (3.4 mL)was added dropwise, followed by 15% aqueous sodium hydroxide (3.4 mL),and finally water (10.2 mL). The resultant slurry was warmed to roomtemperature, stirred for 15 mins, and magnesium sulfate was added,stirring for an additional 15 mins. Solid materials were removed byfiltration through a glass fitted filter, washing multiple times withwarm methylene chloride, and the organic solution was concentrated underreduced pressure to give the desired product as a clear oil (4.5 g,73%). ¹H NMR (300 MHz, CDCl₃): δ 3.05-2.96 (quint, 1H, J=6.6 Hz),2.74-2.71 (t, 2H, J=6.6 Hz), 2.68-2.58 (t, 2H, J=6.9 Hz), 1.85-1.68 (m,2H), 1.62-1.42 (m, 6H), 1.34 (br s, 3H), 1.30-1.21 (m, 2H).

Preparation 26:1-((3-(cyclohexylamino)propyl)amino)-3-(3,6-difluoro-9H-carbazol-9-yl)-2-methylpropan-2-ol;General procedure

To a solution of a N-cyclohexyl-1,3-propanediamine (or otherN-functionalized 1,3-propanediamine, 8.0 equiv.) in ethanol (1 M) wasadded 3,6-difluoro-9-((2-methyloxiran-2-yl)methyl)-9H-carbazole (1.0 eq,or alternatively 3,6-difluoro-9-(oxiran-2-ylmethyl)-9H-carbazole) andthe reaction mixture was stirred at 70° C. for 16 hrs or until reactionwas determined complete by LCMS, cooled to room temperature,concentrated in vacuo to give a crude residue, which was purified bycolumn chromatography, eluting from HP silica gel with an appropriategradient of methanol in methylene chloride and 0.1% triethylamine togive the desired product.

Structure Name Characterization

1-((3- (cyclohexylamino)propyl) amino)-3-(3,6-difluoro-9H-carbazol-9-yl)-2- methylpropan-2-ol ¹H NMR (300 MHz, CDCl₃): δ7.63-7.60 (dd, 2H, J = 2.6, 8.6 Hz), 7.49-7.45 (dd, 2H, J = 4.2, 9.0Hz), 7.20-7.13 (td, 2H, J = 2.4, 9.0 Hz), 2.68-2.53 (m, 6H), 2.36- 2.29(m, 1H), 1.84-1.50 (m, 6H), 1.30- 0.94 (m, 6H), 1.23 (s, 3H); ESI (m/z):430.3 (M + H).

1-((2-aminoethyl)amino)- 3-(3,6-difluoro-9H- carbazol-9-yl)propan-2-ol¹H NMR (300 MHz, CDCl₃): δ 7.68-7.64 (dd, 2H, J = 2.3, 9.0 Hz),7.41-7.37 (dd, 2H, J = 4.0, 9.0 Hz), 7.23-7.16 (td, 2H, J = 2.4, 9.0Hz), 4.33-4.31 (d, 2H, J = 5.7 Hz), 4.16-4.09, (m, 1H), 2.80-2.70 (m,3H), 2.63-2.55 (m, 3H), 1.74 (br s, 4H); ESI (m/z): 320.1 (M + H); HPLCanalysis: (C18, 10-90% acetonitrile in water + 0.1% trifluoroacetic acidover 20 min: retention time, % area at 254 nm): 7.39 min, 97%.

1-(3,6-difluoro-9H- carbazol-9-yl)-3-((3- (phenylamino)propyl)amino)propan-2-ol ¹H NMR (300 MHz, CDCl₃): δ 7.65-7.62 (dd, 2H, J = 2.7,8.7 Hz), 7.45-7.41 (dd, 2H, J = 4.2, 9.0 Hz), 7.25-7.13 (m, 3H),6.72-6.66 (tt, 1H, J = 1.1, 8.7 Hz), 6.59- 6.55 (m, 2H), 4.34-4.32 (d,2H, J = 5.4 Hz), 4.17-4.11 (m, 1H), 3.17-3.13 (t, 2H, J = 6.8 Hz),2.81-2.56 (m, 6H), 1.79-1.70 (m, 2H); ESI (m/z): 410.2 (M + H).

1-((3- (cyclohexylamino)propyl) amino)-3-(3,6-difluoro-9H-carbazol-9-yl)propan- 2-ol: ¹H NMR (300 MHz, CDCl₃): δ 7.66-7.62 (dd,2H, J = 2.7, 8.7 Hz), 7.40-7.35 (dd, 2H, J = 4.2, 9.0 Hz), 7.22-7.15(td, 2H, J = 2.5, 9.0 Hz), 4.29-4.27 (d, 2H, J = 5.4 Hz), 4.11-4.06 (m,1H), 2.72-2.62 (dd, 1H, J = 3.6, 12.0 Hz), 2.68-2.47 (m, 5H), 2.35-2.26(tt, 1H, J = 3.6, 10.7 Hz), 1.80- 1.49 (m, 7H), 1.26-0.93 (m, 7H).

1-((3- (cyclobutylamino)propyl) amino)-3-(3,6-difluoro-9H-carbazol-9-yl)propan- 2-ol ¹H NMR (300 MHz, CDCl₃): δ 7.65-7.61 (dd,2H, J = 2.6, 8.6 Hz), 7.39-7.34 (dd, 2H, J = 4.2, 8.7 Hz), 7.22-7.14 (m,2H), 4.27-4.21 (m, 2H), 4.10-4.03 (m, 1H), 3.14-1.44 (m, 10H).

1-(3,6-difluoro-9H- carbazol-9-yl)-2-methyl- 3-((3- (phenylamino)propyl)amino)propan-2-ol ¹H NMR (300 MHz, CDCl₃): δ 7.66-7.63 (dd, 2H, J = 2.6,8.9 Hz), 7.50-7.45 (dd, 2H, J = 4.2, 9.0 Hz), 7.22-7.14 (m, 4H),6.73-6.67 (tt, 1H, J = 1.0, 7.4 Hz), 6.57- 6.54 (m, 2H), 4.27 (d, 2H, J= 1.5 Hz), 3.12-3.07 (t, 2H, J = 6.8 Hz), 2.73-2.63 (m, 5H), 1.79-1.67(quint, 2H, J = 6.8 Hz), 1.28 (s, 3H); ESI (m/z): 424.2 (M + H).

1-(3,6-difluoro-9H- carbazol-9-yl)-3-((3- (isopropylamino)propyl)amino)-2-methylpropan- 2-ol ¹H NMR (300 MHz, CDCl₃): δ 7.64-7.60 (dd,2H, J = 2.6, 8.6 Hz), 7.48-7.44 (dd, 2H, J = 4.2, 9.0 Hz), 7.19-7.13(td, 2H, J = 2.6, 9.0 Hz), 4.20 (s, 2H), 2.78-2.51 (m, 8H), 1.62-1.53(quint, 2H, J = 6.7 Hz), 1.22 (s, 3H), 1.05-1.03 (d, 3H, J = 6.3 Hz),1.04-1.02 (d, 3H, J = 6.3 Hz); ESI (m/z): 390.2 (M + H).

1-((3- (cyclobutylamino)propyl) amino)-3-(3,6-difluoro-9H-carbazol-9-yl)-2- methylpropan-2-ol ¹H NMR (300 MHz, CDCl₃): δ7.66-7.62 (dd, 2H, J = 2.6, 9.0 Hz), 7.55-7.51 (dd, 2H, J = 4.2, 9.0Hz), 7.23-7.16 (td, 2H, J = 2.6, 9.0 Hz), 4.38-4.27 (m, 2H), 3.38- 3.29(quint, 1H, J = 8.0 Hz), 3.00-2.96 (d, 1H, J = 12.3 Hz), 2.86-2.71 (m,3H), 2.24-1.66 (m, 6H), 1.28 (s, 3H); ESI (m/z): 402.2 (M + H).

1-(3,6-difluoro-9H- carbazol-9-yl)-3-((3- (isopropylamino)propyl)amino)propan-2-ol ¹H NMR (300 MHz, CDCl₃) δ 7.66-7.62 (dd, 2H, J = 2.7,8.7 Hz), 7.39-7.35 (dd, 2H, J = 4.2, 9.0 Hz), 7.21-7.15 (td, 2H, J =2.5, 9.0 Hz), 4.29-4.27 (d, 2H, J = 5.4 Hz), 4.12-4.04 (m, 1H),2.75-2.44 (m, 8H), 1.57-1.48 (quint, 2H, J = 6.9 Hz), 1.01-0.98 (d, 6H,J = 6.3 Hz); ESI (m/z): 376.2 (M + H).

1-((2-aminoethyl)amino)- 3-(9H-carbazol-9- yl)propan-2-ol ¹H NMR (300MHz, CDCl₃): δ 8.09 (m, 2H), 7.50-7.42 (m, 4H), 7.25-7.20 (m, 2H),4.44-4.35 (m, 2H), 4.22-4.14 (m, 1H), 2.78-2.53 (m, 7H), 1.64 (br s,3H); ESI (m/z): 284.1 (M + H); HPLC analysis: (C18, 10-90% acetonitrilein water + 0.1% trifluoroacetic acid over 20 min: retention time, % areaat 254 nm): 6.5 min, 94%.

Preparation 27: tert-butyl (1-amino-1-oxopropan-2-yl)carbamate

A mixture of Boc-DL-alanine (5.0 g, 26.4 mmol, 1.0 equiv.),N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uroniumhexafluorophosphate (15.033 g, 39.6 mmol, 1.5 equiv.),N,N-diisopropylethylamine (8.735 mL, 52.9 mmol, 2.0 equiv.) andanhydrous dimethylformamide (50 mL) was stirred at room temperature for20 mins, and then cooled with ice-water and ammonia (2.250 g, 132.1mmol, 5.0 equiv.) was slowly bubbled into the mixture. The reaction wasstirred at room temperature for 3 hrs in a sealed vessel. The reactionwas diluted with water and extracted with ethyl acetate. The organiclayers were washed with saturated aqueous sodium chloride, dried overanhydrous sodium sulfate, filtered and concentrated in vacuo. Theobtained solids were washed with cold ethyl acetate and ether and driedto afford the product as a white powder (2.9 g, 58%). ¹H NMR (300 MHz,CDCl₃): δ 6.20 (br s, 1H), 5.50 (br s, 1H), 5.00 (br s, 1H), 4.20 (m,1H), 1.47 (s, 9H), 1.40 (d, 3H, J=7.2 Hz).

Preparation 28: tert-butyl (1-aminopropan-2-yl)carbamate

tert-butyl (1-amino-1-oxopropan-2-yl)carbamate (2.2 g) was dissolved inanhydrous tetrahydrofuran (100 mL) and borane (40 mL of 1 M solution intetrahydrofuran) was added. The mixture was stirred at room temperaturefor 2 hrs and then heated at 90° C. for 2 hrs. After cooling to roomtemperature, the reaction was quenched with methanol until no bubbleswere generated. The mixture was heated at 90° C. for 1 hr and thenconcentrated down to dryness to afford the crude product as a syrup (2.2g), which was used directly for the next step reaction. ¹H NMR (300 MHz,CDCl₃): δ 4.60 (br s, 1H), 3.65 (m, 1H), 2.76 (dd, 1H, J=12.9, 5.1 Hz),2.64 (dd, 1H, J=12.9, 6.3 Hz), 1.47 (s, 9H), 1.14 (d, 3H, J=6.9 Hz).

Preparation 29: tert-butyl(1-((3-(3,6-difluoro-9H-carbazol-9-yl)-2-hydroxypropyl)amino)propan-2-yl)carbamate

Under a nitrogen atmosphere, a solution of3,6-difluoro-9-(oxiran-2-ylmethyl)-9H-carbazole (0.7 g) and tert-butyl(1-aminopropan-2-yl)carbamate (1.5 g) in ethanol (50 mL) was stirred at70° C. for 16 hrs. The mixture was concentrated under reduced pressureand purified by silica gel column chromatography, eluting with agradient of 0-20% methanol in methylene chloride to give an off-whitefoam (1.28 g). The product was used directly in the next step withoutadditional purification: ¹H NMR (300 MHz, CDCl₃): δ 7.67 (dd, 2H, J=8.7,2.7 Hz), 7.42-7.37 (m, 2H), 7.22 (td, 2H, J=9.0, 2.7 Hz), 4.55-4.30 (m,3H), 4.13 (m, 1H), 3.78 (br s, 1H), 2.88 and 2.82 (dd, 1H, J=12.0, 3.6Hz), 2.70-2.50 (m, 3H), 1.45 (s, 9H), 1.13 and 1.11 (d, 3H, J=6.6 Hz);ESI (m/z): 434.0 (M+H).

Preparation 30: 3-(cyclopropylamino)propanenitrile

Cyclopropylamine (4.214 mL, 60.8 mmol, 1.0 equiv.) was added slowly to asolution of acrylonitrile (4.840 g, 91.2 mmol, 1.5 equiv.) in methanol(7 mL) at room temperature and stirred for 30 mins. The reaction washeated to reflux and stirred for 1 hour, cooled, concentrated anddistilled under vacuum to give 5.5 g of clear liquid (5.5 g, 82%). ¹HNMR (300 MHz; CDCl₃): δ 2.99 (t, 2H, J=6.3 Hz), 2.51 (t, 2H, J=6.3 Hz),2.12 (m, 1H), 1.78 (br s, 1H), 0.49-0.32 (m, 4H); ESI (m/z): 111.5(M+H).

Preparation 31: N1-cyclopropylpropane-1,3-diamine

To a cooled (0° C.) suspension of lithium aluminum hydride (3.445 g,90.8 mmol, 2.0 equiv.) in anhydrous tetrahydrofuran (120 mL) was slowlyadded over ten minutes a solution of 3-(cyclopropylamino)propanenitrile(5.000 g, 45.4 mmol, 1.0 equiv.) in anhydrous tetrahydrofuran (20 mL).The reaction was stirred at room temperature for 15 mins, than heated toreflux and stirred for 3 hours. The mixture was cooled to roomtemperature and sodium sulfate decahydrate was added until foamingstopped. The suspension was stirred for 10 mins and the solids filteredoff (washing with tetrahydrofuran). The solution was concentrated underreduced pressure to give a crude product, which was used directly in thenext step. ¹H NMR (300 MHz, CDCl₃): δ 2.74-2.69 (m, 4H), 2.10-2.04 (m,1H), 1.65-1.56 (m, 2H), 0.43-0.27 (m, 4H); ESI (m/z): 115.4 (M+H).

Preparation 32: 1-ethyltetrahydropyrimidin-2(1H)-one; General method forsynthesis of ureas from diamines

With vigorous stirring, the appropriate N-functionalized1,3-propanediamine or 1,2-ethylenediamine (10.0 mmol, 1.0 equiv.) wasadded to a solution of 1,1′-carbonyldiimidazole (1.622 g, 10.0 mmol, 1.0equiv.) in anhydrous tetrahydrofuran (0.05 M), which was kept at 0° C.with an external ice bath. The solution was allowed to slowly warm toroom temperature and stirred for 16 hrs. The mixture was worked up byeither: i) the mixture was concentrated under reduced pressure andpurified by column chromatography eluting from silica gel with agradient of methanol in methylene chloride to give the desired product;or ii) the mixture was diluted with ethyl acetate and successivelywashed twice with 1 N aqueous hydrochloric acid and once with saturatedaqueous sodium chloride, back-extracting organic layer once with ethylacetate, dried combined organic fractions over anhydrous sodium sulfate,filtered, and concentrated in vacuo to afford the desired product, whichwas used without further purification.

Structure Name Characterization

1-ethyltetrahydropyrimidin-2(1H)- one ¹H NMR (300 MHz, CDCl₃): δ 4.74(br s, 1H), 3.40-3.33 (q, 2H, J = 6.9 Hz), 3.29- 3.23 (m, 4H), 1.97-1.89(quin, 2H, J = 5.9 Hz), 1.14-1.09 (t, 3H, J = 7.2 Hz).

1-ethylimidazolidin-2-one ¹H NMR (300 MHz, CDCl₃): δ 3.44 (br s, 4H),3.28-3.21 (q, 2H, J = 7.4 Hz), 2.08 (s 1H), 1.15-1.10 (t, 3H, J = 7.1Hz).

1-cyclohexylimidazolidin-2-one ¹H NMR (300 MHz, CDCl₃): δ 4.47 (s, 1H),3.73-3.66 (m, 1H), 3.44-3.35 (m, 4H), 1.81-1.63 (m, 5H), 1.45-1.26 (m,4H), 1.14-1.04 (m, 1H).

1-phenylimidazolidin-2-one ¹H NMR (300 MHz, CDCl₃): δ 7.55-7.52 (m, 2H),7.37-7.30 (m, 2H), 7.08-7.02 (m, 1H), 4.88 (s, 1H), 3.98-3.93 (m, 2H),3.61-3.56 (t, 2H, J = 7.7 Hz).

1-isopropylimidazolidin-2-one ¹H NMR (300 MHz, CDCl₃): δ 4.70 (s, 1H),4.20-4.07 (sept, 1H, J = 6.9 Hz), 3.40-3.35 (m, 4H), 1.14-1.12 (d, 6H, J= 6.9 Hz).

1-cyclopentylimidazolidin-2-one ¹H NMR (300 MHz, CDCl₃): δ 4.45 (s, 1H),4.32-4.24 (m, 1H), 3.42-3.41 (m, 4H), 1.87-1.77 (m, 2H), 1.71-1.45 (m,6H).

1-cyclopropylimidazolidin-2-one ¹H NMR (300 MHz, CDCl₃): δ 4.72 (s, 1H),3.39-3.32 (m, 4H), 2.51-2.36 (m, 1H), 0.76-0.63 (m, 4H).

1-cyclobutylimidazolidin-2-one ¹H NMR (300 MHz, CDCl₃): δ 4.80 (s, 1H),4.49-4.37 (m, 1H), 3.53-3.37 (m, 4H), 2.19-2.03 (m, 4H), 1.69-1.60 (m,2H).

1-cyclopropyltetrahydropyrimidin- 2(1H)-one ¹H NMR (300 MHz, CDCl₃): δ3.57-3.52, 3.06-3.01 (ABq, 2H, J = 14.4 Hz), 3.42 (m, 4H), 2.68-2.67,2.61-2.60 (ABq, 2H, J = 4.5 Hz), 2.43-2.36 (m, 1H), 1.33 (s, 3H),0.74-0.62 (m, 4H); ESI (m/z): 197.1 (M + H).

1-cyclobutyltetrahydropyrimidin- 2(1H)-one ¹H NMR (300 MHz, CDCl₃): δ4.98-4.87 (quin, 1H, J = 8.9 Hz), 4.76 (br s, 1H), 3.30-3.23 (m, 4H),2.13-2.04 (m, 4H), 1.96-1.88 (m, 2H), 1.66-1.58 (m, 2H).

Preparation 31:1-ethyl-3-(oxiran-2-ylmethyl)tetrahydropyrimidin-2(1H)-one; Generalmethod for preparation of (oxiran-2-ylmethyl)-functionalized ureas

To a solution of 1-ethyltetrahydropyrimidin-2(1H)-one, or alternativecyclic urea generated in Preparation 6 (1.0 equiv.), in anhydroustetrandrofuran (0.2 M) was added 60% sodium hydride in mineral oil (1.1equiv.) and the resultant suspension was stirred for 1 hour at roomtemperature. Epibromohydrin (3.0 equiv.) was added and the mixturestirred for 24 hrs at either room temperature or 35° C. Silica gel wasadded and the suspension concentrated under reduced pressure anddirectly purified by silica gel column chromatography—eluting thedesired product with an appropriate gradient of methanol in methylenechloride.

Structure Name Characterization

1-ethyl-3-(oxiran-2- ylmethyl)tetrahydropyrimidin- 2(1H)-one ¹H NMR (300MHz, CDCl₃): δ 3.95-3.90 (m, 1H), 3.44-3.20 (m, 6H), 3.11-3.00 (m, 2H),2.73-2.70 (m, 1H), 2.49-2.47 (m, 1H), 1.96- 1.88 (m, 2H), 1.09-1.04 (m,3H).

1-cyclohexyl-3-(oxiran-2- ylmethyl)imidazolidin-2-one ¹H NMR (300 MHz,CDCl₃): δ 3.78-3.72 (dd, 1H, J = 2.6, 14.6 Hz), 3.71-3.66 (m, 1H),3.50-3.41 (m, 1H), 3.35-3.25 (m, 3H), 3.11- 3.05 (m, 1H), 2.98-2.90 (dd,1H, J = 6.6, 14.7 Hz), 2.78-2.75 (dd, 1H, J = 4.1, 4.5 Hz), 2.57-2.54(dd, 1H, J = 2.3, 5.0 Hz), 1.80-1.62 (m, 5H), 1.40-1.30 (m, 4H),1.09-1.05 (m, 1H).

1-(oxiran-2-ylmethyl)-3- phenylimidazolidin-2-one ¹H NMR (300 MHz,CDCl₃): δ 7.56-7.52 (m, 2H), 7.36-7.30 (m, 2H), 7.07-7.01 (m, 1H),3.92-3.79 (m, 3H), 3.73-3.65 (m, 1H), 3.59- 3.46 (m, 1H), 3.18-3.05 (m,2H), 2.83-2.80 (t, 1H, J = 4.2 Hz), 2.63-2.60 (dd, 1H, J = 2.6, 4.7 Hz).

1-isopropyl-3-(oxiran-2- ylmethyl)imidazolidin-2-one ¹H NMR (300 MHz,CDCl₃): δ 4.21-4.08 (sept, 1H, J = 6.8 Hz), 3.78-3.73 (dd, 1H, J = 2.7,14.7 Hz), 3.52-3.24 (m, 4H), 3.11-3.06 (m, 1H), 2.98-2.91 (dd, 1H, J =6.6, 14.7 Hz), 2.79-2.76 (dd, 1H, J = 3.6, 4.8 Hz), 2.57-2.55 (dd, 1H, J= 2.7, 4.5 Hz), 1.47-1.12 (dd, 6H, J = 1.2, 6.9 Hz).

1-cyclopentyl-3-(oxiran-2- ylmethyl)imidazolidin-2-one ¹H NMR (300 MHz,CDCl₃): δ 4.32-4.22 (quint, 1H, J = 8.0 Hz), 3.78-3.72 (dd, 1H, J = 2.7,14.7 Hz), 3.51-3.26 (m, 4H), 3.09-3.06 (m, 1H), 2.97-2.90 (dd, 1H, J =6.5, 14.6 Hz), 2.78-2.75 (t, 1H, J = 4.4 Hz), 2.57-2.54 (dd, 1H, J =2.6, 4.7 Hz), 1.84-1.47 (m, 8H).

1-cyclopropyl-3-(oxiran-2- ylmethyl)imidazolidin-2-one ¹H NMR (300 MHz,CDCl₃): δ 3.76-3.70 (dd, 1H, J = 3.0, 14.7 Hz), 3.47-3.28 (m, 4H),3.09-3.03 (m, 1H), 2.97-2.90 (dd, 1H, J = 6.3, 14.7 Hz), 2.77-2.74 (t,1H, J = 4.5 Hz), 2.56- 2.53 (dd, 1H, J = 2.7, 4.8 Hz), 2.43-2.36 (m,1H), 0.73-0.61 (m, 4H).

1-cyclobutyl-3-(oxiran-2- ylmethyl)imidazolidin-2-one ¹H NMR (300 MHz,CDCl₃): δ 4.50-4.38 (quint, 1H, J = 8.7 Hz), 3.77-3.71 (dd, 1H, J = 2.6,14.6 Hz), 3.51-3.33 (m, 5H), 3.10-3.04 (m, 1H), 2.98-2.91 (dd, 1H, J =6.6, 14.7 Hz), 2.78-2.75 (t, 1H, J = 4.4 Hz), 2.57-2.54 (dd, 1H, J =2.6, 5.0 Hz), 2.18-2.04 (m, 4H), 1.70- 1.59 (m, 2H).

1,5-dimethyl-3-(oxiran-2- ylmethyl)tetrahydropyrimidin- 2(1H)-one ESI(m/z): 185.1 (M + H).

Preparation 32:1-ethyl-3-((2-methyloxiran-2-yl)methyl)tetrahydropyrimidin-2(1H)-one;General method for preparation of N-((2-methyloxiran-2-yl)methyl)functionalized ureas

To a solution of cyclic urea generated in Preparation 7 (1.0 equiv.) inanhydrous tetrandrofuran (0.2 M) was added 60% sodium hydride in mineraloil (1.1 equiv.) and the resultant suspension was stirred for 1 hr atroom temperature. 2-(Chloromethyl)-2-methyloxirane (4.0 equiv.) wasadded at room temperature and the mixture stirred at 70-90° C. in asealed tube overnight. Silica gel was added and the suspensionconcentrated under reduced pressure and directly purified by silica gelcolumn chromatography—eluting the desired product with a gradient ofmethanol in methylene chloride.

Structure Name Characterization

1-ethyl-3-((2-methyloxiran-2- yl)methyl)tetrahydropyrimidin- 2(1H)-one¹H NMR (300 MHz, CDCl₃): δ 3.78-3.73 (d, 1H, J = 14.7 Hz), 3.40-3.24 (m,7H), 2.63-2.61 and 2.59-2.57 (ABq, 2H, J = 4.8 Hz), 1.97-1.89 (quint,2H, J = 5.9 Hz), 1.31 (s, 3H), 1.11-1.08 (m, 3H).

Preparation 33: 3-(methylamino)butanenitrile

A mixture of methylamine (158.584 mL of a 40% w/w aqueous solution,1842.3 mmol, 10.0 equiv.) and crotononitrile (15 mL, 184.2 mmol, 1.0equiv.) was stirred at room temperature for 16 hrs. The reaction wasextracted with methylene chloride (3×100 mL) and the combined organiclayers were dried over anhydrous sodium sulfate, filtered andconcentrated in vacuo to afford the desired product as a colorless oil(18 g, 100%). ¹H NMR (300 MHz, CDCl₃): δ 2.96 (m, 1H), 2.47 (d, 2H,J=6.0 Hz), 2.46 (s, 3H), 1.36 (d, 3H, J=6.6 Hz).

Preparation 34: N3-methylbutane-1,3-diamine

A Parr shaker flask was charged with methanol (100 mL), cooled to 0° C.and bubbled with ammonia. 3-(Methylamino)butanenitrile (4.8 g, 48.9mmol, 1.0 equiv.) and a spoonful of Raney Ni were added. The reactionwas shaken under hydrogen at 50 psi for 10 hrs. The reaction wasfiltered through Celite and the filtrate concentrated in vacuo to afforda clear oil (5.0 g, 100%). ¹H NMR (300 MHz, CDCl₃): δ 2.90-2.60 (m, 3H),2.43 (s, 3H), 1.70-1.40 (m, 2H), 1.08 (d, 3H, J=6.0 Hz).

Preparation 35: 1,6-dimethyltetrahydropyrimidin-2(1H)-one

To a stirred solution of N3-methylbutane-1,3-diamine (5.0 g, 48.9 mmol,1.0 equiv.) in anhydrous tetrahydrofuran (100 mL) at 0° C. was added1,1′-carbonyldiimidazole (7.935 g, 48.9 mmol, 1.0 equiv.). The mixturewas slowly warmed to room temperature and stirred for 16 hrs. Thereaction was concentrated under reduced pressure and the residue treatedwith saturated aqueous ammonium chloride and extracted twice withmethylene chloride. The combined organic layers were washed withsaturated aqueous sodium chloride, dried over anhydrous sodium sulfate,filtered and concentrated in vacuo. The residue was purified by silicagel column (0-20% ethanol/methylene chloride) to afford the desiredproduct as a white solid (1.8 g, 29%). ¹H NMR (300 MHz, CDCl₃): δ 4.66(br s, 1H), 3.53-3.35 (m, 2H), 3.25 (m, 1H), 2.95 (s, 3H), 2.05 (m, 1H),1.70 (m, 1H), 1.24 (d, 3H, J=6.3 Hz).

Preparation 36: 2-(aminomethyl)-N-benzylaniline

Under a nitrogen atmosphere, a solution of 2-(benzylamino)benzonitrile(4.0 g, 19.2 mmol, 1.0 equiv.) in anhydrous tetrahydrofuran (25 mL) wasadded slowly to a cold (0° C.) suspension of lithium aluminum hydride(2.187 g, 57.6 mmol, 3.0 equiv.) in anhydrous tetrahydrofuran (60 mL).The mixture was cooled and sodium sulfate decahydrate was added untilfoaming stopped (cooling with external water/ice bath). The mixture wasfiltered to remove solids and the solution concentrated in vacuo to aclear liquid (3.2 g, 79%). ¹H NMR (300 MHz, CDCl₃): δ 7.42-7.23 (m, 5H),7.17-7.11 (td, 1H, J=1.8, 7.5 Hz), 7.07-7.04 (m, 1H), 6.68-6.61 (m, 2H),6.29 (br s, 1H), 4.40 (s, 2H), 3.99 (s, 2H), 1.31 (br s, 2H).

Preparation 37: 1-benzyl-3,4-dihydroquinazolin-2(1H)-one

To a solution of 2-(aminomethyl)-N-benzylaniline (1.5 g, 7.1 mmol, 1.0equiv.) in anhydrous tetrahydrofuran (142 mL) was added1,1′-carbonyldiimidazole (1.318 g, 8.1 mmol, 1.1 equiv.) and thesolution stirred at room temperature for 24 hrs. 1 N aqueoushydrochloric acid (20 mL) was added and the mixture extracted threetimes with ethyl acetate. The organic fractions were dried withanhydrous sodium sulfate, filtered and concentrated in vacuo to afford awhite solid (1.68 g, 99%). ¹H NMR (300 MHz, CDCl₃): δ 7.33-7.20 (m, 5H),7.11-7.03 (m, 2H), 6.95-6.91 (m, 1H), 6.74-6.71 (d, 1H, J=8.1 Hz), 5.13(s, 2H), 4.54 (s, 2H), 1.60 (br s, 2H). ESI (m/z): 239.2 (M+H).

Preparation 38:1-benzyl-3-(3-(3,6-difluoro-9H-carbazol-9-yl)-2-hydroxypropyl)-3,4-dihydroquinazolin-2(1H)-one

To a stirred solution of 1-benzyl-3,4-dihydroquinazolin-2(1H)-one (0.105g, 0.4 mmol, 1.8 equiv.) in anhydrous dimethylformamide (1 mL) was added60% sodium hydride in mineral oil (0.01 g, 0.3 mmol, 1.0 equiv.) and themixture was stirred for 30 mins.3,6-Difluoro-9-(oxiran-2-ylmethyl)-9H-carbazole (0.065 g, 0.3 mmol, 1.0equiv.) was added and the mixture stirred for 16 hrs at 55° C. Themixture was poured into saturated aqueous ammonium chloride andextracted three times with ethyl acetate. The organic fractions weredried over anhydrous sodium sulfate, filtered and concentrated to afforda tan oil. The crude residue was purified by silica gel columnchromatography, eluting with a gradient of 20-60% ethyl acetate/hexanesto give product that was used directly in the next step withoutadditional purification (35%). ESI (m/z): 498.2 (M+H); HPLC analysis:(C18, 5-95% acetonitrile in water+0.1% trifluoroacetic acid over 20 min:retention time, % area at 254 nm): 15.2 mins, 51%.

Preparation 39:1-benzyl-3-(3-(3,6-difluoro-9H-carbazol-9-yl)-2-hydroxy-2-methylpropyl)-3,4-dihydroquinazolin-2(1H)-one

To a stirred solution of 1-benzyl-3,4-dihydroquinazolin-2(1H)-one (0.099g, 0.4 mmol, 1.8 equiv.) in anhydrous dimethylformamide (1 mL) was added60% sodium hydride in mineral oil (0.01 g, 0.3 mmol, 1.0 equiv.) and themixture stirred for 30 mins.3,6-Difluoro-9-((2-methyloxiran-2-yl)methyl)-9H-carbazole (0.065 g, 0.2mmol, 1.0 equiv.) was added and the mixture stirred at 55° C. for 16hrs. The mixture was poured into saturated aqueous ammonium chloride andextracted three times with ethyl acetate. The organic fractions weredried over sodium sulfate, filtered and concentrated to afford a tanoil. The crude residue was purified by silica gel column chromatography,eluting with a gradient of 20-60% ethyl acetate/hexanes to give anoff-white solid (0.086 g, 65%). ¹H NMR (300 MHz, CDCl₃): δ 7.66-7.62(dd, 2H, J=2.4, 8.7 Hz), 7.45-7.40 (dd, 2H, J=4.1, 8.9 Hz), 7.30-7.09(m, 8H), 7.04-6.93 (m, 2H), 6.75-6.72 (d, 1H, J=8.1 Hz), 5.20-5.15 and5.08-5.03 (ABq, 2H, J=16.6 Hz), 4.74-4.69 and 4.59-4.54 (ABq, 2H, J=14.3Hz), 4.39-4.34 and 4.32-4.26 (ABq, 2H, J=15.3 Hz), 4.09 (s, 1H),3.97-3.93 and 3.51-3.47 (ABq, 2H, J=14.4 Hz), 1.36 (s, 3H); ESI (m/z):512.3 (M+H).

Preparation 40: (R)-(2-methyloxiran-2-yl)methanol

A mixture of crushed 4 Å activated molecular sieves (15.0 g) andmethylene chloride (300 mL) was cooled to −10° C., titanium(IV)isopropoxide (2.072 mL, 7.0 mmol, 0.05 equiv.) and(−)-diethyl-D-tartrate (2.126 g, 10.3 mmol, 0.07 equiv.) were added bysyringe followed by 80% cumene hydroperoxide (50.000 g, 262.8 mmol, 1.8equiv.). The mixture was stirred at −10° C. for 30 mins. The mixture wascooled to −35° C. and a solution of 2-methyl-2-propen-1-ol (10.620 g,147.3 mmol, 1.0 equiv.) in dichloromethans (10 mL) was added by syringe,in 1-2 mL portions over 30 mins. The reaction mixture was stirred at−35° C. for 1 h and then placed in a −20° C. freezer for 3 days. Themixture was warmed to 0° C., water was added and stirred for 30 mins atroom temperature. The mixture was cooled to 0° C. and a solution of 30%sodium hydroxide in saturated aqueous sodium chloride (10 mL) was addedand stirred at this temperature for 1 h. The mixture was filteredthrough a pad of Celite and washed with methylene chloride (50 mL). Theaqueous portion was separated and extracted with methylene chloride(3×30 mL). The organic layer was combined and dried (anhydrous magnesiumsulfate), filtered and concentrated in vacuo to a colorless liquid. Thecrude residue was purified by silica gel column chromatography, elutingwith a gradient of 20-100% diethyl ether/hexanes to give a colorless oil(6.2349 g 48%). ¹H NMR (300 MHz, CDCl₃): δ 3.76 (dd, 1H, J=4.5, 12.3Hz), 3.6 (dd, 1H, J=8.1, 12.3 Hz), 2.92 (d, 1H, J=4.5 Hz), 2.66 (d, 1H,J=4.8 Hz), 1.79 (br m, 1H), 1.36 (s, 3H).

The (S) enantiomer was synthesized in a similar manner from the(+)-diethyl tartrate.

Preparation 41: (S)-(2-methyloxiran-2-yl)methyl 3-nitrobenzenesulfonate

To a stirred solution of (R)-(2-methyloxiran-2-yl)methanol (5.000 g,56.8 mmol, 1.0 equiv.), N,N-4-dimethylaminopyridine (0.100 g, 0.8 mmol,1.4 mol %) and N,N-diisopropylethylamine (15.776 mL, 88.9 mmol, 2.0equiv.) in anhydrous methylene chloride (100 mL) at −20° C. was slowlyadded 3-nitrobenzenesulfonyl chloride (15.092 g, 68.1 mmol, 1.2 equiv.)in small portions over 15 mins. The mixture was allowed to reach 0° C.and stirred for 3 h. The reaction was quenched with water and extractedwith methylene chloride. The combined organic layers were washedsequentially with water, 1 N aqueous hydrochloric acid, saturatedaqueous sodium bicarbonate, and saturated aqueous sodium chloride. Theorganic layer was dried (anhydrous sodium sulfate), filtered andconcentrated in vacuo. The crude residue was purified by silica gelcolumn (0-80% ethyl acetate/hexanea) to afford the desired product as ayellow oil (6.73 g, 43.4%). ¹H NMR (300 MHz, CDCl₃): δ 8.77 (t, 1H,J=1.8 Hz), 8.54 (m, 1H), 8.27 (m, 1H), 7.84 (t, 1H, J=7.95 Hz), 4.29 (d,1H, J=11.1 Hz), 4.05 (d, 1H, J=11.1 Hz), 2.73 (dd, 2H, J=17.7, 4.8 Hz),1.37 (s, 3H).

The (R) enantiomer was synthesized in a similar manner.

Preparation 42: (S)-9-(oxiran-2-ylmethyl)-9H-carbazole

To a stirred solution of carbazole (5.15 g, 30.8 mmol, 1.0 equiv.) inanhydrous dimethylformamide (100 mL) at 0° C. was added 60% sodiumhydride in mineral oil (1.355 g, 33.9 mmol, 1.1 equiv.) and the mixturewas stirred at 0° C. for 1 hr. (R)-(−)-glycidyl nosylate (9.981 g, 38.5mmol, 1.3 equiv.) was added and the reaction mixture stirred for 1 hrand then slowly warmed to room temperature and stirred for 16 hrs. Themixture was partitioned between water and ethyl acetate. The organiclayer was washed with saturated aqueous sodium chloride, dried(anhydrous sodium sulfate), filtered and concentrated under reducedpressure to give a red oil. The crude residue was purified by silica gelcolumn (15-80% methylene chloride/hexanes) to afford the desired productas a white solid (4.95 g, 72%). ¹H NMR (300 MHz, CDCl₃): δ 8.11-8.08(dt, 2H, J=0.9, 7.5 Hz), 7.48-7.46 (m, 4H), 7.28-7.23 (m, 2H), 4.67-4.61(dd, 1H, J=2.4, 15.9 Hz), 4.45-4.38 (dd, 1H, J=5.0, 15.9 Hz), 3.39-3.34(m, 1H), 2.83-2.80 (t, 1H, J=4.2 Hz), 2.60-2.57 (dd, 1H, J=2.4, 4.8 Hz).HPLC analysis: (C18, 5-95% acetonitrile in water+0.1% trifluoroaceticacid over 20 min: retention time, % area at 254 nm): 13.2 mins, 99.5%.Chiral HPLC analysis: (Chiralcel AD-H, 5-15% isopropanol in hexanes over20 mins: retention time, % area at 254 nm): 8.12 mins, 3.8%; 8.54 mins,96.1% (92.2% ee).

The following compounds were prepared analogously:

Structure Name Characterization

(R)-9-(oxiran-2- ylmethyl)-9H-carbazole ¹H NMR (300 MHz, CDCl₃): δ8.11-8.08 (dt, 2H, J = 0.9, 7.5 Hz), 7.48-7.46 (m, 4H), 7.28-7.23 (m,2H), 4.67- 4.61 (dd, 1H, J = 2.4, 15.9 Hz), 4.45-4.38 (dd, 1H, J = 5.0,15.9 Hz), 3.39-3.34 (m, 1H), 2.83-2.80 (t, 1H, J = 4.2 Hz), 2.60-2.57(dd, 1H, J = 2.4, 4.8 Hz); HPLC analysis: (C18, 5-95% acetonitrile inwater + 0.1% trifluoroacetic acid over 20 min: retention time, % area at254 nm): 13.2 mins, 99.4%; Chiral HPLC analysis: (Chiralcel AD-H, 5-15%isopropanol in hexanes over 20 mins: retention time, % area at 254 nm):8.13 mins, 94.9%; 8.55 mins, 5.0% (89.9% ee).

(S)-3,6-difluoro-9- (oxiran-2-ylmethyl)-9H- carbazole ¹H NMR (300 MHz,CDCl₃): δ 7.68-7.65 (dd, 2H, J = 2.6, 8.6 Hz), 7.39-7.35 (dd, 2H, J =4.2, 9.0 Hz), 7.24- 7.18 (td, 2H, J = 2.6, 9.0 Hz), 4.69-4.62 (dd, 1H, J= 2.9, 5.0 Hz), 4.33-4.26 (dd, 1H, J = 5.0, 16.1 Hz), 3.35-3.31 (m, 1H),2.84-2.81 (t, 1H, J = 4.2 Hz), 2.54-2.52 (dd, 1H, J = 2.6, 4.7 Hz); HPLCanalysis: (C18, 5-95% acetonitrile in water + 0.1% trifluoroacetic acidover 20 min: retention time, % area at 254 nm): 13.8 mins, 100%; ChiralHPLC analysis: (Chiralcel OD-H, 25% isopropanol in hexanes over 20 mins:retention time, % area at 254 nm): 7.7 mins, 1.8%; 10.1 mins, 98.2%(96.4% ee).

(R)-3,6-difluoro-9- (oxiran-2-ylmethyl)-9H- carbazole ¹H NMR (300 MHz,CDCl₃): δ 7.68-7.65 (dd, 2H, J = 2.6, 8.6 Hz), 7.39-7.35 (dd, 2H, J =4.2, 9.0 Hz), 7.24- 7.18 (td, 2H, J = 2.6, 9.0 Hz), 4.69-4.62 (dd, 1H, J= 2.9, 5.0 Hz), 4.33-4.26 (dd, 1H, J = 5.0, 16.1 Hz), 3.35-3.31 (m, 1H),2.84-2.81 (t, 1H, J = 4.2 Hz), 2.54-2.52 (dd, 1H, J = 2.3, 4.7 Hz); HPLCanalysis: (C18, 5-95% acetonitrile in water + 0.1% trifluoroacetic acidover 20 min: retention time, % area at 254 nm): 13.7 mins, 100%; ChiralHPLC analysis: (Chiralcel OD-H, 25% isopropanol in hexanes over 20 mins:retention time, % area at 254 nm): 7.7 mins, 98.8%; 10.2 mins, 1.2%(97.5% ee).

(R)-9-((2-methyloxiran-2- yl)methyl)-9H-carbazole ¹H NMR (300 MHz,CDCl₃): δ 8.09-8.12 (m, 2H), 7.49 (d, 4H, J =3.9 Hz), 7.23-7.28 (m,2H),4.63 (d, 1H, J = 15.9 Hz), 4.32 (d, 1H, J = 15.9 Hz), 2.69 (s, 2H),1.33 (s, 3H); HPLC analysis: (C18, 10-90% acetonitrile in water + 0.1%trifluoroacetic acid over 20 min: retention time, % area at 254 nm):13.65 min, 89.2%; Chiral HPLC analysis (Phenomenex Lux 3 μ cellulose-2,4% isopropanol in hexanes over 20 mins: retention time, % area at 254nm): 6.94 min, 90.0%, 7.83 min, 10.0% (80.0% ee).

(S)-9-((2-methyloxiran-2- yl)methyl)-9H-carbazole ¹H NMR (300 MHz,CDCl₃): δ 8.09-8.12 (m, 2H), 7.49 (d, 4H, J = 3.9 Hz), 7.23-7.28 (m,2H), 4.63 (d, 1H, J = 15.9 Hz), 4.32 (d, 1H, J = 15.9 Hz), 2.69 (s, 2H),1.33 (s, 3H); HPLC analysis: (C18, 10-90% acetonitrile in water + 0.1%)trifluoroacetic acid over 20 min: retention time, % area at 254 nm):13.68 min, 100%; Chiral HPLC analysis (Phenomenex Lux 3 μ cellulose-2,4%) isopropanol in hexanes over 20 mins: retention time, % area at 254nm): 6.95 min, 7.1%, 7.83 min, 92.9% (85.8% ee).

(R)-3,6-difluoro-9-((2- methyloxiran-2- yl)methyl)-9H-carbazole ¹H NMR(300 MHz, CDCl₃): δ 7.69 (dd, 2H, J = 9.0, 2.4 Hz), 7.42 (dd, 2H, J =8.7, 3.9 Hz), 7.25 (td, 2H, J = 9.0, 2.4 Hz), 4.63 (d, 1H, J = 15.9 Hz),4.23 (d, 1H, J = 15.9 Hz), 2.69 (dd, 2H, J = 15.3, 4.8 Hz), 1.31 (s,3H); HPLC analysis: (C18, 10-90% acetonitrile in water + 0.1%trifluoroacetic acid over 20 min: retention time, % area at 254 nm):14.1 min, 98.4%,; Chiral HPLC analysis: (Chiralcel AD-H, 4% ethanol inhexanes over 20 mins: retention time, % area at 254 nm): 11.7 mins,95.4%; 15.7 mins, 4.6% (90.8% ee).

(S)-3,6-difluoro-9-((2- methyloxiran-2- yl)methyl)-9H-carbazole ¹H NMR(300 MHz, CDCl₃): δ 7.69 (dd, 2H, J = 9.0, 2.4 Hz), 7.42 (dd, 2H, J =8.7, 3.9 Hz), 7.25 (td, 2H, J = 9.0, 2.4 Hz), 4.63 (d, 1H, J = 15.9 Hz),4.23 (d, 1H, J = 15.9 Hz), 2.69 (dd, 2H, J = 15.3, 4.8 Hz), 1.31 (s,3H); HPLC analysis: (C18, 10-90% acetonitrile in water + 0.1%trifluoroacetic acid over 20 min: retention time, % area at 254 nm):14.1 min, 100%; Chiral HPLC analysis: (Chiralcel AD-H, 4% ethanol inhexanes over 20 mins: retention time, % area at 254 nm): 11.7 mins,4.4%; 15.9 mins, 95.6% (91.2% ee).

Preparation 43: (1S,4R)-2-azabicyclo[2.2.1]heptan-3-one

A mixture of (1R)-(−)-2-azabicyclo[2.2.1]hept-5-en-3-one (1.0 g, 9.2mmol) and 10% palladium on carbon (0.4 g) in methanol (50 mL) wasstirred under an atmosphere of hydrogen for 3 hrs. The mixture wasfiltered through Celite and the filter cake washed with methanol. Thecombined organics were concentrated in vacuo to afford the requireproduct (1 g, 98%). ¹H NMR (300 MHz, CDCl₃): δ 5.91 (br s, 1H), 3.89 (s,1H), 2.74 (s, 1H), 1.96-1.59 (m, 5H), 1.42-1.37 (m, 1H).

Preparation 44: (1R,4S)-2-azabicyclo[2.2.1]heptan-3-one

A mixture of (1S)-(+)-2-azabicyclo[2.2.1]hept-5-en-one (1.0 g, 9.2 mmol,1.0 equiv.) and 10% palladium on carbon (0.4 g) in methanol (40 mL) wasstirred under an atmosphere of hydrogen at ambient temperature for 2hrs. The mixture was filtered through Celite and the filter cake washedwith methanol. The combined organics were concentrated in vacuo toafford the required product (1 g, 98%). ¹H NMR (300 MHz, CDCl₃): δ 5.83(br s, 1H), 3.89 (s, 1H), 2.74 (s, 1H), 1.96-1.59 (m, 5H), 1.42-1.38 (m,1H).

Preparation 45: (R)-5-(hydroxymethyl)-2-pyrrolidinone p-toluenesulfonate

(R)-(−)-5-(hydroxymethyl)-2-pyrrolidinone (1.0 g, 8.7 mmol, 1.0 equiv.)was dissolved in methylene chloride (40 mL). Triethylamine (1.569 mL,11.3 mmol, 1.3 equiv.), p-toluenesulfonyl chloride (1.904 g, 10.0 mmol,1.1 equiv.) and 4-(dimethylamino)pyridine (0.12 g) were added underice-cooling, and the mixture stirred at room temperature for 18 hrs. Thereaction mixture was concentrated under reduced pressure, 0.5 N aqueoushydrochloric acid was added, and the mixture extracted with ethylacetate. The organic layer was washed with 0.5 N aqueous hydrochloricacid, water, saturated aqueous sodium hydrogen carbonate, and saturatedaqueous sodium chloride. The organic fraction was dried (anhydroussodium sulfate), filtered and concentrated under reduced pressure toafford a white solid (1.91 g, 81%). ¹H NMR (300 MHz, CDCl₃): δ 7.79-7.76(m, 2H), 7.38-7.35 (m, 2H), 5.73 (br s, 1H), 4.08-4.04 (dd, 1H, J=3.5,9.5 Hz), 3.98-3.93 (m, 1H), 3.85-3.82 (dd, 1H, J=7.5, 9.5 Hz), 2.47 (s,3H), 2.36-2.22 (m, 3H), 1.81-1.73 (m, 1H).

Preparation 46: (S)-5-(iodomethyl)pyrrolidin-2-one

(S)-(5-oxopyrrolidin-2-yl)methyl 4-methylbenzenesulfonate (4.5 g, 16.7mmol, 1.0 equiv.) was dissolved in anhydrous acetonitrile (140 mL),sodium iodide (5.009 g, 33.4 mmol, 2.0 equiv.) was added, and themixture heated to reflux for 8 hrs. The reaction mixture was cooled toroom temperature, concentrated under reduced pressure, water added, andthe mixture extracted with ethyl acetate. The organic layer was washedwith saturated aqueous sodium thiosulfate, water, and saturated aqueoussodium chloride. The organic fraction was dried (anhydrous sodiumsulfate), filtered and concentrated in vacuo to give the title compoundas a white solid (2.0 g, 53%). ¹H NMR (300 MHz, CDCl₃): δ 6.42 (br s,1H), 3.90-3.82 (s, 1H), 3.27-3.16 (m, 2H), 2.53-2.29 (m, 3H), 1.88-1.17(m, 1H).

Preparation 47: (R)-5-(iodomethyl)pyrrolidin-2-one

(R)-5-(hydroxymethyl)-2-pyrrolidinone p-toluenesulfonate (1.9 g, 7.1mmol, 1.0 equiv.) was dissolved in anhydrous acetonitrile (60 mL),sodium iodide (2.09 g) was added, and the mixture heated at reflux for 8hrs. The reaction mixture was cooled to room temperature, concentratedunder reduced pressure, water added, and the mixture extracted withethyl acetate. The organic layer was washed with saturated aqueoussodium thiosulfate, water and saturated aqueous sodium chloride. Theorganic fraction was dried (anhydrous sodium sulfate), filtered andconcentrated in vacuo to give the title compound as an off-white solid(0.97 g, 61%). ¹H NMR (300 MHz, CDCl₃): δ 5.92 (br s, 1H), 3.89-3.85 (m,1H), 3.28-3.15 (m, 2H), 2.53-2.30 (m, 3H), 1.88-1.78 (m, 1H).

Preparation 48: (R)-5-methylpyrrolidin-2-one

(S)-5-(Iodomethyl)pyrrolidin-2-one (2.0 g, 8.9 mmol, 1.0 equiv.) wasdissolved in ethanol (60 mL), sodium carbonate (1.036 g, 9.8 mmol, 1.1equiv.) and 10% palladium on carbon (0.4 g) were added, and the mixturestirred for 16 hrs under a hydrogen atmosphere. The mixture was filteredthrough Celite and the filtrate concentrated under reduced pressure toafford an orange semi-solid. To the obtained residue was added 5%aqueous sodium thiosulfate, and the mixture extracted with ethylacetate. The organic layer was dried (anhydrous sodium sulfate),filtered and concentrated in vacuo to give the title compound as a paleyellow oil (0.564 g, 64%). ¹H NMR (300 MHz, CDCl₃): δ 6.61 (br s, 1H),3.82-3.72 (sext, 1H, J=6.6 Hz), 2.45-2.22 (m, 3H), 1.71-1.59 (m, 1H),1.23-1.21 (d, 3H, J=6.6 Hz).

Preparation 49: (S)-5-methylpyrrolidin-2-one

(S)-5-Iodomethylpyrrolidin-2-one (1.12 g) was dissolved in ethanol (30mL), sodium carbonate (0.53 g) and 10% palladium on carbon (0.22 g) wereadded, and the mixture was stirred for 8 hrs under a hydrogenatmosphere. The mixture was filtered through Celite, and the filtrateconcentrated under reduced pressure. To the obtained residue was added5% aqueous sodium thiosulfate, and the mixture extracted with ethylacetate. The organic layer was dried (anhydrous sodium sulfate),filtered and concentrated in vacuo to give the title compound (0.263 g,61%). ¹H NMR (300 MHz, CDCl₃): δ 6.34 (br s, 1H), 3.83-3.72 (sext, 1H,J=6.3 Hz), 2.46-2.21 (m, 3H), 1.72-1.60 (m, 1H), 1.23-1.21 (d, 3H, J=6.0Hz).

Preparation 50:(S)-1-((2-aminoethyl)amino)-3-(3,6-difluoro-9H-carbazol-9-yl)propan-2-ol

A mixture of (R)-3,6-difluoro-9-(oxiran-2-ylmethyl)-9H-carbazole (0.1 g,0.4 mmol, 1.0 equiv.) and ethylenediamine (0.232 g, 3.9 mmol, 10.0equiv.) in ethanol (2 mL) was stirred at 55° C. for 5 hrs. The reactionmixture was cooled to room temperature, and concentrated in vacuo toafford a light yellow syrup which slowly solidified to form an off-whitesolid (0.122 g, 96%). ¹H NMR (300 MHz, CDCl₃): δ 7.68 (dd, 2H, J=8.7,2.7 Hz), 7.42 (dd, 2H, J=8.7, 4.2 Hz), 7.22 (td, 2H, J=9.0, 2.7 Hz),4.33-4.31 (d, 2H, J=5.4 Hz), 4.15 (m, 1H), 2.85-2.55 (m, 6H) 1.84 (br s,4H). ESI (m/z): 320.2 (M+H).

Preparation 51: (R)-4-benzyl-3-(4-bromobutanoyl)oxazolidin-2-one

A round bottomed flask was charged with 4-bromobutyric acid (19.226 g,115.1 mmol, 1.0 equiv.) and dry diethyl ether (500 mL). The resultingsolution was cooled to −78° C., and triethylamine (16.473 mL, 118.5mmol, 1.1 equiv.) was added followed by trimethylacetyl chloride (14.596mL, 118.5 mmol, 1.1 equiv.). A white precipitate formed, upon which timethe cold bath was removed and the reaction mixture allowed to warm to 0°C. and stirred for 2 h, the slurry was the re-cooled to −78° C. In aseparate reaction vessel, 2.5 M n-butyllithium solution in hexanes(45.146 mL, 112.9 mmol, 1.0 equiv.) was added dropwise to a solution of(R)-4-benzyl-2-oxazolidinone (20.000 g, 112.9 mmol, 1.0 equiv.) stirringin anhydrous tetrahydrofuran (170 mL) at −78° C. After 10 min, theresulting slurry was added, by wide-bore cannula, to the mixed anhydride(washed with approx. 100 mL anhydrous tetrahydrofuran). After 15 min at−78° C., the mixture was allowed to warm to 0° C. over 30 min and thenmaintained at this temperature for 1 h. The slurry was then quenchedcarefully with water (180 mL), stirred for an additional 5 min, and thenwarmed to room temperature. The resulting solution was extracted withethyl acetate (2×500 mL). The combined organic extracts were washed withwater, saturated aqueous sodium bicarbonate (500 mL), saturated aqueoussodium chloride (400 mL), dried (anhydrous sodium sulfate), filtered,and concentrated in vacuo. The crude residue was purified by flashchromatography (silica gel, 10-50% ethyl acetate/hexanes) to afford aclear oil (26.0 g, 71%). ¹H NMR (300 MHz, CDCl₃): δ 7.19-7.37 (m, 5H),4.63-4.71 (m, 1H), 4.16-4.25 (m, 2H), 3.52 (t, 2H, J=6.5 Hz), 3.29 (dd,1H, J=3.3, 13.5 Hz), 3.08-3.18 (m, 2H), 2.78 (dd, 1H, J=9.6, 13.5 Hz),2.21-2.30 (m, 2H).

The (S) enantiomer was synthesized in a similar manner.

Preparation 52: (R)-3-(4-azidobutanoyl)-4-benzyloxazolidin-2-one

Sodium azide (7.623 g, 117.3 mmol, 1.5 equiv.) was added to stirredsolution of (R)-4-benzyl-3-(4-bromobutanoyl)oxazolidin-2-one (25.500 g,78.2 mmol, 1.0 equiv.) in anhydrous N,N-dimethylformamide (225 mL). Theresulting solution was heated at 55° C. for 15 min and 2 h at 70° C.After cooling to room temperature, the solution was poured intosaturated aqueous sodium chloride/ethyl acetate (1.4 L) and washed withwater and saturated aqueous sodium chloride, dried (anhydrous sodiumsulfate), filtered, and concentrated in vacuo to give an oil (22.0 g,98%), which was used without further purification. ¹H NMR (300 MHz,CDCl₃): δ 7.36-7.19 (m, 5H), 4.62-4.72 (m, 1H), 4.10-4.25 (m, 2H), 3.41(t, 2H, J=6.6 Hz), 3.30 (dd, 1H, J=3.3, 13.2 Hz), 2.99-3.10 (m, 2H),2.79 (dd, 2H, J=9.9, 13.5 Hz), 1.93-2.02 (m, 2H); HPLC analysis: (C18,10-90% acetonitrile in water+0.1% trifluoroacetic acid over 20 min:retention time, % area at 254 nm): 12.3 min, 95%.

The (S) enantiomer was synthesized in a similar manner.

Preparation 53:(R)-3-((R)-4-azido-2-methylbutanoyl)-4-benzyloxazolidin-2-one

To a solution of (R)-3-(4-azidobutanoyl)-4-benzyloxazolidin-2-one(20.600 g, 71.5 mmol, 1.0 equiv.) stirring in anhydrous tetrahydrofuranat −78° C. (125 mL) was slowly added 1.0 M sodiumbis(trimethylsilyl)amide solution in tetrahydrofuran (119.088 mL, 71.5mmol, 1.0 equiv.). After 15 min, the resulting solution was treated withiodomethane (4.671 mL, 75.0 mmol, 1.0 equiv.). The cooling bath wasremoved, and after 5 min the reaction vessel was placed in an ice-waterbath and stirred for an additional 15 min. The reaction was quenchedwith saturated aqueous sodium bicarbonate (300 mL) and extracted withethyl acetate (2×300 mL). The combined organic extracts were washed withsaturated aqueous sodium chloride (2×200 mL), dried (anhydrous magnesiumsulfate), filtered, and concentrated in vacuo. Purification by flashchromatography (400 g silica gel, 10-50% ethyl acetate/hexanes) affordedthe desired product as a clear oil (9.7 g, 45%). ¹H NMR (300 MHz,CDCl₃): δ 7.20-7.38 (m, 5H), 4.65-4.72 (m, 1H), 4.15-4.26 (m, 2H),3.85-3.79 (m, 1H), 3.35 (app. t, 2H, J=6.0 Hz), 3.25 (dd, 1H, J=13.5,3.6 Hz), 2.78 (dd, 1H, J=13.0 Hz, 9.0 Hz), 2.04-2.18 (m, 1H), 1.67-1.78(m, 1H), 1.27 (d, 3H, J=6.9 Hz); HPLC analysis: (C18, 10-90%acetonitrile in water+0.1% trifluoroacetic acid over 20 min: retentiontime, % area at 254 nm): 13.0 min, 99.4%.

The (S) enantiomer was synthesized in a similar manner.

Preparation 54: (R)-3-methylpyrrolidin-2-one

To a solution of(R)-3-((R)-4-azido-2-methylbutanoyl)-4-benzyloxazolidin-2-one (9.650 g,31.9 mmol, 1.0 equiv.) stirring in anhydrous tetrahydrofuran (125 mL) atroom temperature was added triphenylphosphine (16.744 g, 63.8 mmol, 2.0equiv.). After 5 min (solution turns yellow and bubbles), water (0.575mL, 31.9 mmol, 1.0 equiv.) was added and the reaction mixture wasstirred at 25° C. for 18 h. The mixture was concentrated and theresulting residue purified by flash chromatography (silica gel, 75-100%ethyl acetate/hexanes, then 100% to 90% ethyl acetate/methanol gradient)to afford the desired product (2.98 g, 95%). ¹H NMR (300 MHz, CDCl₃): δ6.10 (br. s, 1H), 3.25-3.35 (m, 2H), 2.27-2.50 (m, 2H), 1.68-1.80 (m,1H), 1.20 (d, 3H, J=7.5 Hz).

The (S) enantiomer was synthesized in a similar manner.

Preparation 55: (S)-3-((trimethylsilyl)oxy)pyrrolidin-2-one

Following the procedure of Harris, B. D. et al. Synth. Commun. 1986, 16,1815. Trimethylsilyl chloride (0.805 mL, 6.3 mmol, 0.1 equiv.) was addedto a stirred mixture of (S)-(−)-4-amino-2-hydroxybutyric acid (15.000 g,125.9 mmol, 1.0 equiv.) in xylene (1 L) and hexamethyldisilazane(184.757 mL, 881.5 mmol, 7.0 equiv.) at room temperature. The reactionmixture was heated to reflux for 4 h, cooled to room temperature, anddiluted with absolute ethanol/methanol (1 L). The solvents were removedunder reduced pressure to give the crude product which was purified bycolumn chromatography using ethyl acetate/methylene chloride (30-80%gradient) as eluant to afford a white solid. (17.9 g, 82%). ¹H NMR (300MHz, CDCl₃): δ 6.49 (br s, 1H), 4.28-4.23 (t, 1H, J=7.7 Hz), 3.40-3.21(m, 2H), 2.42-2.32 (m, 2H), 2.08-1.95 (m, 2H), 0.18 (s, 9H).

Preparation 56: (S)-tert-butyl2-oxo-3-((trimethylsilyl)oxy)pyrrolidine-1-carboxylate

Following the procedure of DiRocco, D. A. et al. J. Am. Chem. Soc. 2009,131, 10872 & DiRocco, D. A. et al. WO 2012/009372. To a solution of(S)-3-(trimethylsilyloxy)pyrrolidin-2-one (6.00 g, 34.62 mmol, 1.0equiv) in anhydrous methylene chloride (150 mL) was added di-tert-butyldicarbonate (15.11 g, 69.24 mmol, 2.0 equiv), triethylamine (4.82 mL,34.62 mmol, 1.0 equiv), and 4-dimethylaminopyridine (4.23 g, 34.62 mmol,1.0 equiv). The mixture was stirred overnight at room temperature, then1 N aqueous hydrochloric acid (100 mL) was added, and the layersseparated. The organic layer was washed with 1 N aqueous hydrochloricacid (2×50 mL), and saturated aqueous sodium chloride (1×50 mL), dried(anhydrous sodium sulfate) and filtered. The solution was concentratedin vacuo to afford a crude oil, which was purified by silica gelchromatography (eluting with 0-15% ethyl acetate/hexanes) to afford aclear viscous oil, which solidified in freezer. (3.93 g, 42%). ¹H NMR(300 MHz, CDCl₃): δ 4.32-4.26 (dd, 1H, J=8.4, 9.3 Hz), 3.82-3.74 (ddd,1H, J=2.1, 9.0, 11.1 Hz), 3.50-3.41 (m, 1H), 2.28-2.25 (m, 1H),1.94-1.87 (m, 1H), 1.51 (s, 9H), 0.18 (s, 9H).

Preparation 57: (R)-3-fluoropyrrolidin-2-one

A solution of (S)-tert-butyl2-oxo-3-((trimethylsilyl)oxy)pyrrolidine-1-carboxylate (3 g, 11 mmol) inanhydrous methylene chloride (55 mL) was cooled to −78° C. at whichpoint diethylaminosulfur trifluoride (2.9 mL, 22 mmol, 2.0 equiv) wasadded dropwise. The solution was then allowed to slowly warm to roomtemperature and saturated aqueous sodium bicarbonate (50 mL) was thenadded to quench the reaction. The layers were separated and the organiclayer was then washed with saturated aqueous ammonium chloride (2×25mL), dried (anhydrous sodium sulfate), filtered, and concentrated invacuo to yield a crude solid. This crude material was then dissolved inmethylene chloride (40 mL) and trifluoroacetic acid (2.5 mL, 33 mmol,3.0 equiv) was added. The solution was stirred for 3 h at which pointthe evolution of gas had subsided. Concentration in vacuo afforded a tanliquid which was purified by silica gel chromatography (eluting with0-10% methanol/methylene chloride) yielding the desired product as awhite solid (0.77 g, 68%). ¹H NMR (300 MHz, CDCl₃): δ 7.35 (br s, 1H),5.15-4.93 (dt, 1H, J=7.6, 53.4 Hz), 3.50-3.34 (m, 2H), 2.53 (m, 1H),2.39-2.21 (m, 1H).

Preparation 58: (R)-3-hydroxypyrrolidin-2-one

To a stirring mixture of 4-nitrobenzoic acid (9.273 g, 55.5 mmol, 1.1equiv.) and (S)-(−)-3-3ydroxy-2-pyrrolidone (5.100 g, 50.4 mmol, 1.0equiv.) in anhydrous tetrahydrofuran (175 mL) under a nitrogenatmosphere, triphenylphosphine (26.461 g, 100.9 mmol, 2.0 equiv.) wasadded. To this reaction mixture, disopropyl azodicarboxylate (14.898 mL,75.7 mmol, 1.5 equiv.) was added dropwise (with external cooling withcold water bath). The reaction was stirred at room temperatureovernight. The reaction mixture was concentrated in vacuo to afford acrude residue. Methanol (130 mL) was added to the residue followed bypotassium carbonate (0.38 g) at room temperature. The reaction mixturewas stirred at room temperature for 8 h. The reaction mixture wasdiluted with methylene chloride and filtered through Celite. The Celitebed was washed with 1% methanol in methylene chloride. The filtrateswere combined and concentrated to dryness. The residue was partitionedbetween ethyl acetate: dilute aqueous hydrochloric acid (20 mL, 9:1) andstirred for 15 min. The layers were separated and the aqueous layerwashed with ethyl acetate three times. The aqueous layer wasconcentrated to dryness and a solid residue was obtained. The cruderesidue was washed with 1-2% methanol in methylene chloride (3×50 mL),dried (anhydrous sodium sulfate), filtered, and concentrated to afford atan oil (3.3 g, 60%). ¹H NMR (300 MHz, CDCl₃): δ 4.32-4.27 (t, 1H, J=8.5Hz), 3.36-3.19 (m, 2H), 2.48-2.40 (m, 1H), 2.07-1.93 (m, 1H), 1.16-1.14(d, 1H, J=6.3 Hz).

Preparation 59: (R)-3-((trimethylsilyl)oxy)pyrrolidin-2-one

Trimethylsilyl chloride (0.405 mL, 3.2 mmol, 0.1 equiv.) was added to astirred suspension of (R)-3-hydroxy-2-pyrrolidone (3.200 g, 31.7 mmol,1.0 equiv.), xylene (45 mL), and hexamethyldisilazane (39.8 mL, 189mmol, 6.0 equiv.) at room temperature. The reaction mixture was heatedat reflux temperature for 5 h, and diluted with absolute ethanol (50mL). The solvents were removed under reduced pressure. The crude productwas purified by column chromatograph using ethyl acetate in methylenechloride (30-80% gradient) as eluant to afford a clear oil thatsolidifies to an off-white solid upon standing. (2.57 g, 47%). ¹H NMR(300 MHz, CDCl₃): δ 6.89 (br s, 1H), 4.28-4.23 (t, 1H, J=8.1 Hz),3.40-3.21 (m, 2H), 2.42-2.32 (m, 1H), 2.08-1.95 (m, 1H), 0.18 (s, 9H).

Preparation 60: (R)-tert-butyl2-oxo-3-((trimethylsilyl)oxy)pyrrolidine-1-carboxylate

To a solution of (R)-3-((trimethylsilyl)oxy)pyrrolidin-2-one (2.770 g,16.0 mmol, 1.0 equiv.) in anhydrous methylene chloride (75 mL) was addeddi-tert-butyl dicarbonate (6.971 g, 31.9 mmol, 2.0 equiv.),triethylamine (2.222 mL, 16.0 mmol, 1.0 equiv.), andN,N-4-dimethylaminopyridine (1.953 g, 16.0 mmol, 1.0 equiv.). Themixture was stirred overnight at room temperature then diluted withmethylene chloride and washed with 0.1 N aqueous hydrochloric acid (100mL). The organic layer was washed with 0.1 N aqueous hydrochloric acid(2×100 mL), and saturated aqueous sodium chloride (1×100 mL), dried(anhydrous sodium sulfate), and filtered. The solution was concentratedin vacuo and purified by silica gel chromatography (eluting with 0-15%ethyl acetate/hexanes) to afford a clear viscous oil (2.1 g, 48%). ¹HNMR (300 MHz, CDCl₃): δ 4.32-4.26 (dd, 1H, J=8.1, 9.3 Hz), 3.82-3.74(ddd, 1H, J=2.1, 9.0, 11.1 Hz), 3.50-3.41 (m, 1H), 2.32-2.23 (m, 1H),1.97-1.87 (m, 1H), 1.54 (s, 9H), 0.18 (s, 9H).

Preparation 61: (S)-3-fluoropyrrolidin-2-one

A solution of (R)-tert-butyl2-oxo-3-((trimethylsilyl)oxy)pyrrolidine-1-carboxylate (2.100 g, 7.7mmol, 1.0 equiv.) in anhydrous methylene chloride (37 mL) was cooled to−78° C. at which point diethylaminosulfur trifluoride (2.030 mL, 15.4mmol, 2.0 equiv.) was added dropwise. The solution was then allowed toslowly warm to room temperature and saturated aqueous sodium bicarbonate(33 mL) was then added to quench the reaction. The layers were separatedand the organic layer washed with saturated aqueous ammonium chloride(2×16 mL), dried (anhydrous sodium sulfate), filtered, and concentratedin vacuo to yield a crude solid. This crude material was then dissolvedin dichloromethane (30 mL) and trifluoroacetic acid (1.7 mL, 33 mmol,3.0 equiv) was added. The solution was stirred for 3 h at which pointthe evolution of gas had subsided. Concentration in vacuo andpurification by silica gel chromatography (eluting with 0-10%methanol/methylene chloride) yielded the desired product as an off-whitesolid (0.71 g, 90%). ¹H NMR (300 MHz, CDCl₃): δ 7.35 (br s, 1H),5.15-4.93 (dt, 1H, J=7.6, 53.4 Hz), 3.50-3.34 (m, 2H), 2.53 (m, 1H),2.39-2.21 (m, 1H).

Preparation 62: methyl 4-methyl-5-oxopentanoate

Following the procedure of Oikawa, M. et al. Tetrahedron, 1995, 51,62377. A flask containing piperidine (54.425 mL, 551.0 mmol, 2.0 equiv.)and potassium carbonate (13.774 g, 99.7 mmol, 0.4 equiv.) was immersedin a water bath, and propionaldehyde (16.000 g, 275.5 mmol, 1.0 equiv.)was added over 20 min with vigorous stirring. After being stirred for 18h, the insoluble material was removed by filtering through a pad ofCelite (pad washed with diethyl ether). The filtrate was dried(anhydrous sodium sulfate), filtered and concentrated in vacuo. Thecrude enamine thus obtained was dissolved in acetonitrile (150 mL), andto this was added methyl acylate (47.433 g, 551.0 mmol, 2.0 equiv.)dropwise. The reaction mixture was stirred at reflux for 24 h, followedby the addition of acetic acid (31.541 mL, 551.0 mmol, 2.0 equiv.) andwater (150 mL). After being stirred at reflux for 24 h, it was saturatedwith sodium chloride and extracted with diethyl ether (3×100 mL). Thecombined organic extracts were dried (anhydrous magnesium sulfate),filtered, and concentrated in vacuo. Purification by silica gel flashchromatography (0-20% ethyl acetate/hexanes) afforded the adduct as acolorless oil (22 g, 55%). ¹H NMR (300 MHz, CDCl₃): δ 9.64 (d, 1H, J=1.8Hz), 3.69 (s, 3H), 2.50-2.35 (m, 3H), 2.07 (sext, 1H, J=7.2 Hz), 1.71(sext, 1H, J=7.2 Hz), 1.14 (d, 3H, J=7.2 Hz).

Preparation 63:(3R,8S,8aR)-8-methyl-3-phenyltetrahydro-2H-oxazolo[3,2-a]pyridin-5(3H)-oneand(3R,8R,8aS)-8-methyl-3-phenyltetrahydro-2H-oxazolo[3,2-a]pyridin-5(3H)-one

Following the procedure of Amat, M. et al. J. Org. Chem. 2014, 79, 2792.To a stirred solution of methyl 4-methyl-5-oxopentanoate (5.780 g, 40.1mmol, 1.0 equiv.) in toluene (100 mL) was added (R)-(−)-phenylglycinol(5.500 g, 40.1 mmol, 1.0 equiv.). The reaction mixture was heated atreflux for 25 h with azeotropic removal of the produced water withDean-Stark apparatus. After cooling, the mixture was concentrated invacuo and the residue was purified by silica gel column (column waspre-treated with TEA, then eluted with 0-70% ethyl acetate/hexanes) toafford the desired products:(3R,8S,8aS)-8-methyl-3-phenyltetrahydro-2H-oxazolo[3,2-a]pyridin-5(3H)-oneas a light yellow solid (1.3 g, 14%) and(3R,8S,8aR)-8-methyl-3-phenyltetrahydro-2H-oxazolo[3,2-a]pyridin-5(3H)-oneas a colorless syrup (6.5 g, 70%).(3R,8S,8aS)-8-methyl-3-phenyltetrahydro-2H-oxazolo[3,2-a]pyridin-5(3H)-one: ¹H NMR (300 MHz, CDCl₃): δ 7.40-7.20 (m, 5H), 5.25 (t, 1H,J=7.5 Hz), 4.61 (d, 1H, J=8.4 Hz), 4.48 (t, 1H, J=8.7 Hz), 3.75 (dd, 1H,J=9.0, 7.8 Hz), 2.55 (dd, 1H, J=18.0, 6.0 Hz), 2.46-2.33 (m, 1H),1.88-1.51 (m, 3H), 1.19 (d, 3H, J=5.7 Hz); ESI (m/z): 232.7 (M+H).(3R,8S,8aR)-8-methyl-3-phenyltetrahydro-2H-oxazolo[3,2-a]pyridin-5(3H)-one: ¹H NMR (300 MHz, CDCl₃): δ 7.40-7.20 (m, 5H), 4.93 (d, 1H,J=6.3 Hz), 4.44 (d, 1H, J=9.3 Hz), 4.14 (dd, 1H, J=8.7, 6.3 Hz), 4.01(dd, 1H, J=8.7, 1.2 Hz), 2.47-2.25 (m, 2H), 2.05-1.87 (m, 2H), 1.60-1.43(m, 1H), 1.20 (d, 3H, J=6.6 Hz); ESI (m/z): 232.7 (M+H).

Preparation 64:(S)-1-((R)-2-hydroxy-1-phenylethyl)-5-methylpiperidin-2-one

Following the procedure of Amat, M. et al. J. Org. Chem. 2014, 79, 2792.To a stirred solution of(3R,8S,8aR)-8-methyl-3-phenyltetrahydro-2H-oxazolo[3,2-a]pyridin-5(3H)-one (3.600 g, 15.6 mmol, 1.0 equiv.) in anhydrous methylenechloride (100 mL) was added triethylsilane (7.458 mL, 46.7 mmol, 3.0equiv.) and titanium (IV) chloride (7.697 mL, 70.0 mmol, 4.5 equiv.),and the mixture was stirred at 50° C. for 24 h. Then, additionaltitanium (IV) chloride (7.7 mL) and triethylsilane (7.5 mL) were added,and the stirring was continued at 50° C. for 24 h. The mixture waspoured into saturated aqueous sodium bicarbonate (100 mL). The aqueousphase was filtered over Celite and extracted with methylene chloride.The combined organic extracts were dried (anhydrous sodium sulfate),filtered, and concentrated in vacuo to afford a residue which waschromatographed over silica gel (0-100% ethyl acetate/hexanes, then pureethyl acetate) to afford the desired product as a colorless oil (1.6 g,44%) and starting material (0.6 g) was recovered. ¹H NMR (300 MHz,CDCl₃): δ 7.40-7.20 (m, 5H), 5.80 (dd, 1H, J=9.6, 4.8 Hz), 4.20-4.00 (m,2H), 2.97 (ddd, 1H, J=11.7, 4.8, 2.4 Hz), 2.84 (dd, 1H, J=11.7, 9.9 Hz),2.70 (br s, 1H), 2.59 (ddd, 1H, J=17.7, 6.3, 3.0 Hz), 2.47 (ddd, 1H,J=17.7, 11.1, 6.6 Hz), 1.90-1.75 (m, 2H), 1.50 (m, 1H), 0.93 (d, 3H,J=6.3 Hz).

Preparation 65: (S)-5-methylpiperidin-2-one

In a two-necked flask, fitted with a dry ice condenser, was charged(S)-1-((R)-2-hydroxy-1-phenylethyl)-5-methylpiperidin-2-one (1.600 g,6.9 mmol, 1.0 equiv.) and anhydrous tetrandrofuran (20 mL). The mixturewas cooled to −78° C. under a nitrogen atmosphere, and then ammonia (50mL) was condensed. The reaction temperature was raised to −33° C. Smallpieces of metal sodium were added until the blue color persisted, andthe mixture was stirred at −33° C. for 5 min. The reaction was quenchedby addition of solid ammonium chloride until the blue color disappeared.The mixture was stirred at room temperature for 5 h and methylenechloride was added. The mixture was filtered through Celite and thefiltrate concentrated in vacuo. The residue was purified by silica gelcolumn (0-100% ethyl acetate/hexanes and then 20% methanol/ethylacetate) to afford the desired product as a colorless oil (0.480 g,62%). ¹H NMR (300 MHz, CDCl₃): δ 5.84 (br s, 1H), 3.10 (m, 1H), 2.94 (t,1H, J=10.8 Hz), 2.50-2.28 (m, 2H), 2.05-1.80 (m, 2H), 1.50 (m, 1H), 1.03(d, 3H, J=6.9 Hz).

Preparation 66:(R)-1-((R)-2-hydroxy-1-phenylethyl)-5-methylpiperidin-2-one

Following the procedure of Amat, M. et al. J. Org. Chem. 2014, 79, 2792.To a stirred solution of(3R,8R,8aS)-8-methyl-3-phenyltetrahydro-2H-oxazolo[3,2-a]pyridin-5(3H)-one (1.000 g, 4.3 mmol, 1.0 equiv.) in anhydrous methylene chloride(50 mL) was added triethylsilane (2.072 mL, 13.0 mmol, 3.0 equiv.) andtitanium (IV) chloride (2.138 mL, 19.5 mmol, 4.5 equiv.), and themixture was stirred at 50° C. for 6 h. The mixture was poured intosaturated aqueous sodium bicarbonate (50 mL). The aqueous phase wasfiltered over Celite and extracted with methylene chloride. The combinedorganic extracts were dried (anhydrous sodium sulfate), filtered, andconcentrated in vacuo to afford a residue, which was chromatographed(0-100% ethyl acetate/hexanes and then ethyl acetate) to afford thedesired product as a colorless syrup (0.200 g, 20%). ¹H NMR (300 MHz,CDCl₃): δ 7.40-7.20 (m, 5H), 5.67 (dd, 1H, J=7.8, 6.3 Hz), 4.20-4.05 (m,2H), 3.15-3.00 (m, 2H), 2.65-2.40 (m, 3H), 2.00-1.75 (m, 2H), 1.40 (m,1H), 0.89 (d, 3H, J=6.9 Hz).

Preparation 67: (R)-5-methylpiperidin-2-one

In a two-necked flask, fitted with a dry ice condenser, was charged(R)-1-((S)-2-hydroxy-1-phenylethyl)-5-methylpiperidin-2-one (0.200 g,0.9 mmol, 1.0 equiv.) and anhydrous tetrahydrofuran (5 mL). The mixturewas cooled to −78° C. under a nitrogen atmosphere, and then ammonia (15mL) was condensed. The reaction temperature was raised to −33° C. Smallpieces of metal sodium was added until the blue color persisted, and themixture was stirred at −33° C. for 5 min. The reaction was quenched byaddition of solid ammonium chloride until the blue color disappeared.The mixture was stirred at room temperature for 5 h and methylenechloride was added. The mixture was filtered through Celite and thefiltrate concentrated in vacuo. The residue was purified by silica gelcolumn (0-100% ethyl acetate/hexanes and then 20% methanol/ethylacetate) to afford the desired product as a yellow oil (0.080 g, 82%).¹H NMR (300 MHz, CDCl₃): δ 5.85 (br s, 1H), 3.10 (m, 1H), 2.93 (t, 1H,J=10.8 Hz), 2.50-2.28 (m, 2H), 2.05-1.80 (m, 2H), 1.50 (m, 1H), 1.03 (d,3H, J=6.6 Hz).

Preparation 68: (S)-tert-butyl (1-amino-1-oxopropan-2-yl)carbamate

A mixture of Boc-Ala-OMe (5.000 g, 24.6 mmol, 1.0 equiv.) and 28%aqueous ammonium hydroxide (100.00 mL, 1479.7 mmol, 60.1 equiv.) inmethanol (100 mL) was stirred for 16 h at room temperature. The reactionmixture was concentrated in vacuo to afford the desired product as awhite solid (4.65 g, 100%). ¹H NMR (300 MHz, CDCl₃): δ 6.15 (br, 1H),5.55 (br, 1H), 4.95 (br, 1H), 4.20 (br, 1H), 1.46 (s, 9H), 1.39 (d, 3H,J=7.2 Hz).

Preparation 69: (S)-tert-butyl (1-aminopropan-2-yl)carbamate

To a stirred solution of Boc-Ala-NH₂ (4.600 g, 24.4 mmol, 1.0 equiv.) inanhydrous tetrahydrofuran (100 mL) under nitrogen was added 1.0 Mborane-tetrahydrofuran complex in tetrahydrofuran (85.536 mL, 85.5 mmol,3.5 equiv.). The mixture was stirred for 16 h at room temperature andthen heated at 70° C. for 2 h. After cooling, the reaction was quenchedwith methanol until no bubbles generated. The mixture was heated at 70°C. for 2 h and then concentrated in vacuo. The residue was purified bysilica gel column (0-100% ethyl acetate/hexanes and then 0-30%methanol/methylene chloride) to afford the desired product as asemi-solid (1.4 g, 33%). ¹H NMR (300 MHz, CDCl₃): δ 4.60 (br s, 1H),3.65 (m, 1H), 2.76 (dd, 1H, J=12.9, 4.8 Hz), 2.64 (dd, 1H, J=12.9, 6.6Hz), 1.45 (s, 9H), 1.13 (d, 3H, J=6.9 Hz).

Preparation 70: tert-butyl((S)-1-((S)-3-(3,6-difluoro-9H-carbazol-9-yl)-2-hydroxypropyl)amino)propan-2-yl)carbamate

A mixture of (R)-3,6-difluoro-9-(oxiran-2-ylmethyl)-9H-carbazole (0.600g, 2.3 mmol, 1.0 equiv.) and(S)-tert-butyl-(1-aminopropan-2-yl)carbamate (1.210 g, 6.9 mmol, 3.0equiv.) in ethanol (10 mL) was stirred at 70° C. After 15 h, thereaction mixture was concentrated in vacuo and purified by silica gelcolumn (0-100% ethyl acetate/hexanes then 0-30% methanol/methylenechloride) to afford the desired product as a white foam (0.750 g, 75%)and recovered amine starting material (0.2 g). ¹H NMR (300 MHz, CDCl₃):δ 7.67 (dd, 2H, J=8.7, 2.4 Hz), 7.42 (dd, 2H, J=8.7, 3.9 Hz), 7.22 (td,2H, J=9.0, 2.7 Hz), 4.50-4.25 (m, 3H), 4.13 (m, 1H), 3.78 (br s, 1H),2.86 (dd, 1H, J=12.0, 3.6 Hz), 2.70-2.50 (m, 3H), 1.43 (s, 9H), 1.10 (d,3H, J=6.3 Hz); ESI (m/z): 434.0 (M+H).

Preparation 71: tert-butyl((S)-1-(((S)-3-(9H-carbazol-9-yl)-2-hydroxypropyl)amino)propan-2-yl)carbamate

A mixture of (R)-9-(oxiran-2-ylmethyl)-9H-carbazole (0.150 g, 0.7 mmol,1.0 equiv.) and (S)-tert-butyl (1-aminopropan-2-yl)carbamate (0.200 g,1.1 mmol, 1.7 equiv.) in ethanol (5 mL) was stirred at 70° C. After 15h, the reaction mixture was concentrated in vacuo and purified by silicagel column (0-100% ethyl acetate/hexanes) to afford the desired productas a white foam (0.120 g, 45%). ¹H NMR (300 MHz, CDCl₃): δ 8.09 (d, 2H,J=7.8 Hz), 7.52-7.41 (m, 4H), 7.25-7.20 (m, 2H), 4.50-4.35 (m, 3H), 4.17(m, 1H), 3.73 (m, 1H), 2.83 (dd, 1H, J=11.7, 3.6 Hz), 2.66-2.47 (m, 3H),1.43 (s, 9H), 1.08 (d, 3H, J=6.6 Hz); ESI (m/z): 398.1 (M+H).

Preparation 72: (R)-tert-butyl (1-amino-1-oxopropan-2-yl)carbamate

A mixture of Boc-D-Ala-OMe (5.000 g, 24.6 mmol, 1.0 equiv.) and 28%aqueous ammonium hydroxide (100 mL, 1479.7 mmol, 60.1 equiv.) inmethanol (100 mL) was stirred for 16 h at room temperature. The reactionmixture was concentrated in vacuo to afford the desired product as awhite solid (4.65 g, 100%). ¹H NMR (300 MHz, CDCl₃): δ 6.15 (br, 1H),5.60 (br, 1H), 5.00 (br, 1H), 4.20 (br, 1H), 1.46 (s, 9H), 1.39 (d, 3H,J=6.9 Hz).

Preparation 73: (R)-tert-butyl (1-aminopropan-2-yl)carbamate

To a stirred solution of Boc-D-Ala-NH₂ (4.600 g, 24.4 mmol, 1.0 equiv.)in anhydrous tetrahydrofuran (100 mL) under nitrogen was added 1.0 Mborane-tetrahydrofuran complex in tetrahydrofuran (97.756 mL, 97.8 mmol,4.0 equiv.). The mixture was stirred for 16 h at room temperature andthen heated at 70° C. for 2 h. After cooling, the reaction was quenchedwith methanol until no bubbles generated. The mixture was heated at 70°C. for 2 h and then concentrated in vacuo. The residue was purified bysilica gel column (0-30% methanol/methylene chloride) to afford thedesired product as a colorless oil (1.5 g, 35%). ¹H NMR (300 MHz,CDCl₃): δ 4.60 (br s, 1H), 3.65 (m, 1H), 2.75 (dd, 1H, J=12.9, 5.1 Hz),2.63 (dd, 1H, J=12.9, 6.6 Hz), 1.45 (s, 9H), 1.41 (s, 2H), 1.12 (d, 3H,J=6.3 Hz).

Preparation 74: tert-butyl((R)-1-(((S)-3-(3,6-difluoro-9H-carbazol-9-yl)-2-hydroxypropyl)amino)propan-2-yl)carbamate

A mixture of (R)-3,6-difluoro-9-(oxiran-2-ylmethyl)-9H-carbazole (0.200g, 0.8 mmol, 1.0 equiv.) and (R)-tert-butyl(1-aminopropan-2-yl)carbamate (0.403 g, 2.3 mmol, 3.0 equiv.) in ethanol(10 mL) was stirred at 70° C. After 15 h, the reaction mixture wasconcentrated in vacuo and purified by silica gel column (0-30%methanol/methylene chloride) to isolate the desired product as a whitepowder (0.250 g, 75%). ¹H NMR (300 MHz, CDCl₃): δ 7.68 (dd, 2H, J=8.7,2.7 Hz), 7.41 (dd, 2H, J=9.3, 4.2 Hz), 7.22 (td, 2H, J=9.3, 2.4 Hz),4.43 (br s, 1H), 4.35 (d, 2H, J=5.7 Hz), 4.12 (m, 1H), 3.78 (br s, 1H),2.80 (dd, 1H, J=12.3, 3.6 Hz), 2.63 (dd, 1H, J=12.3, 5.7 Hz), 2.57 (d,2H, J=7.2 Hz), 1.44 (s, 9H), 1.11 (d, 3H, J=6.6 Hz); ESI (m/z): 434.0(M+H).

Preparation 75: tert-butyl((R)-1-(((S)-3-(9H-carbazol-9-yl)-2-hydroxypropyl)amino)propan-2-yl)carbamate

A mixture of (R)-9-(oxiran-2-ylmethyl)-9H-carbazole (0.150 g, 0.7 mmol,1.0 equiv.) and (R)-tert-butyl (1-aminopropan-2-yl)carbamate (0.351 g,2.0 mmol, 3.0 equiv.) in ethanol (10 mL) was stirred at 70° C. After 15h, the reaction mixture was concentrated in vacuo and purified by silicagel column (0-30% methanol/methylene chloride) to isolate the desiredproduct as a white foam (0.210 g, 78%). ¹H NMR (300 MHz, CDCl₃): δ 8.10(d, 2H, J=7.5 Hz), 7.55-7.40 (m, 4H), 7.27-7.20 (m, 2H), 4.60-4.30 (m,3H), 4.16 (m, 1H), 3.78 (m, 1H), 2.80 (dd, 1H, J=12.3, 3.6 Hz), 2.67(dd, 1H, J=12.0, 8.4 Hz), 2.60-2.50 (m, 2H), 1.44 (s, 9H), 1.10 (d, 3H,J=6.6 Hz); ESI (m/z): 398.1 (M+H).

Example 2 Preparation of Compounds of Formula I Compound 1:1-(3-(9H-carbazol-9-yl)-2-hydroxypropyl)piperidin-2-one

To a stirred solution of piperidin-2-one (0.133 g, 1.3 mmol) in dimethylsulfoxide (5 mL) was added potassium tert-butoxide (0.151 g, 1.3 mmol)and the mixture was stirred at room temperature for 1 hour.9-(oxiran-2-ylmethyl)-9H-carbazole (0.150 g, 0.7 mmol) was added and themixture was stirred at room temperature for 16 hours. The mixture wasdiluted with water and extracted with ethyl acetate. The organic layerwas washed with saturated aqueous sodium chloride, dried (anhydroussodium sulfate), filtered and concentrated in vacuo. The residue waspurified by silica gel column (50-100% ethyl acetate/hexanes) and thenby prep-HPLC (C18, 30-80% acetonitrile/water with 0.1% formic acid over0-8 min) to afford the product as a white foam (0.098 g, 45%). ¹H NMR(300 MHz, CDCl₃): δ 8.08 (d, 2H, J=7.8 Hz), 7.50-7.40 (m, 4H), 7.28-7.18(m, 2H), 4.60-4.25 (m, 4H), 3.87 (dd, 1H, J=14.1, 7.8 Hz), 3.15-2.95 (m,3H), 2.36 (t, 2H, J=5.7 Hz), 1.85-1.55 (m, 4H); ESI (m/z): 323.2 (M+H).

Compounds 2 to 72

Compounds 2 to 72 were prepared by procedures analogous to those usedfor Compound 1 or by using sodium hydride (0.4 equiv.) instead ofpotassium tert-butoxide. Alternatively, phosphazene base P4-t-Bu can beused instead of potassium tert-butoxide also. In cases where thestarting materials for the targets below were not commerciallyavailable, the syntheses are described in the preceding preparationexamples.

ESI Cpd Structure Name ¹H NMR (m/z) 2

1-(3-(9H-carbazol- 9-yl)-2- hydroxypropyl)-3- methyltetrahydro-pyrimidin-2(1H)-one (300 MHz, CDCl₃) δ 8.10 (d, 2H, J = 7.8 Hz),7.55-7.40 (m, 4H), 7.32- 7.20 (m, 2H), 5.31 (s, 1H), 4.55-4.25 (m, 3H),3.88 (dd, 1H, J = 14.4, 8.4 Hz), 3.30-2.85 (m, 8H), 2.00- 1.80 (m, 2H).338.2 (M + H) 3

1-(3-(9H-carbazol- 9-yl)-2- hydroxypropyl) tetrahydropyrimidin-2(1H)-one (300 MHz, CDCl₃) δ 8.10 (d, 2H, J = 7.5 Hz), 7.54-7.42 (m,4H), 7.32- 7.20 (m,2H), 5.10 (s, 1H), 4.77 (s, 1H), 4.50-4.28 (m, 3H),3.87 (dd, 1H, J = 14.7, 8.1 Hz), 3.35-3.20 (m, 2H), 3.15-2.90 (m, 3H),1.92-1.80 (m, 2H). 324.2 (M + H) 4

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxypropyl) piperidin-2-one(300 MHz, CDCl₃) δ 7.68 (dd, 2H, J = 8.4, 2.7 Hz), 7.39 (dd, 2H, J =9.0, 4.2 Hz), 7.23 (td, 2H, J = 9.0, 2.7 Hz), 4.50-4.25 (m, 4H), 3.91(dd, 1H, J = 14.4, 8.1 Hz), 3.25-3.08 (m, 2H), 3.02 (d, 1H, J = 14.1Hz), 2.50- 2.38 (m, 2H), 1.90-1.65 (m, 4H). 359.1 (M + H) 5

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxy-2- methylpropyl)piperidin-2-one (300 MHz, CDCl₃) δ 7.67 (dd, 2H, J = 8.7, 2.7 Hz), 7.46(dd, 2H, J = 9.0, 4.2 Hz), 7.21 (td, 2H, J = 9.0, 2.7 Hz), 4.88 (s, 1H),4.30 (s, 2H), 3.99 (d, 1H, J = 14.1 Hz), 3.55-3.30 (m, 2H), 3.17 (d, 1H,J = 14.4 Hz), 2.47 (m, 2H), 1.95-1.70 (m, 4H), 1.30 (s, 3H). 373.0 (M +H) 6

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxypropyl)-3-methyltetrahydro- pyrimidin-2(1H)-one (300 MHz, CDCl₃) δ 7.68 (dd, 2H, J= 8.7, 2.7 Hz), 7.40 (dd, 2H, J = 8.7, 4.2 Hz), 7.22 (td, 2H, J = 8.7,2.7 Hz), 5.27 (br s, 1H), 4.50-4.20 (m, 3H), 3.82 (m, 1H), 3.32-3.00 (m,4H), 2.95 (s, 3H), 2.92 (dd, 1H, J = 14.4, 1.5 Hz), 1.91 (5 peaks, 2H, J= 6.0 Hz). 374.1 (M + H) 7

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxypropyl) pyrrolidin-2-one(300 MHz, CDCl₃) δ 7.68 (dd, 2H, J = 8.4, 2.7 Hz), 7.37 (dd, 2H, J =8.7, 4.2 Hz), 7.23 (td, 2H, J = 9.0, 2.7 Hz), 4.45-4.25 (m, 3H), 3.89(br s, 1H), 3.60-3.25 (m, 4H), 2.45 (t, 2H, J = 8.4 Hz), 2.15-1.95 (m,2H). 345.0 (M + H) 8

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxy-2- methylpropyl)pyrrolidin-2-one (300 MHz, CDCl₃) δ 7.67 (dd, 2H, J = 8.7, 2.7 Hz), 7.44(dd, 2H, J = 9.0, 4.2 Hz), 7.21 (td, 2H, J = 9.0, 2.7 Hz), 4.34 and 4.28(AB, 2H, J = 15.3 Hz), 3.70-3.50 (m, 4H), 3.38 (d, 1H, J = 14.1 Hz),2.47 (t, 2H, J = 8.7 Hz), 2.20-2.00 (m, 2H), 1.29 (s, 3H). 359.0 (M + H)9

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxypropyl)-3-fluoropyrrolidin-2- one (300 MHz, CDCl₃): δ 7.68 (dd, 2H, J = 8.7, 2.7Hz), 7.36 (dd, 2H, J = 8.7, 4.2 Hz), 7.23 (td, 2H, J = 9.0, 2.7 Hz),5.14 (dm, 1H, J = 52.2 Hz), 4.50-4.30 (m, 3H), 3.62-3.30 (m, 4H), 3.12and 3.05 (d, 1H, J = 4.2 Hz), 2.60-2.10 (m, 2H). 363.1 (M + H) 10

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxy-2- methylpropyl)-3-fluoropyrrolidin-2- one (300 MHz, CDCl₃): δ 7.68 (dd, 2H, J = 8.4, 2.7Hz), 7.42 (dd, 2H, J = 9.0, 4.2 Hz), 7.22 (td, 2H, J = 9.0, 2.7 Hz),5.15 (ddd, 1H, J = 52.5, 7.5, 5.7 Hz), 4.40-4.25 (m, 2H), 3.76 (m, 1H),3.65-3.48 (m, 3H), 2.70-2.20 (m, 3H), 1.32 and 1.30 (s, 3H). 377.0 (M +H) 11

1-(3-(3,6-difluoro- 9H-carbonyl-9-yl)-2- hydroxypropyl)-3-fluoropiperidin-2- one (300 MHz, CDCl₃): δ 7.67 (dd, 2H, J = 8.7, 2.7Hz), 7.38 (dd, 2H, J = 9.0, 4.2 Hz), 7.27-7.18 (m, 2H), 4.84 (dm, 1H, J= 47.1 Hz), 4.50-4.25 (m, 3H), 3.80-3.10 (m, 5H), 2.30-1.90 (m, 3H),1.80 (m, 1H). 377.1 (M + H) 12

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxy-2- methylpropyl)-3-fluoropiperidin-2- one (300 MHz, CDCl₃): δ 7.67 (dd, 2H, J = 8.7, 2.7Hz), 7.47-7.40 (m, 2H), 7.21 (td, 2H, J = 9.0, 2.7 Hz), 4.91 (dm, 1H, J= 46.5 Hz), 4.32 (d, 2H, J = 5.4 Hz), 3.87 (dd, 1H, J = 19.8, 13.8 Hz),3.70-3.30 (m, 4H), 2.35- 1.80 (m, 4H), 1.31 (s, 3H). 391.1 (M + H) 13

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2 hydroxypropyl)-3,3-difluoropiperidin-2- one (300 MHz, CDCl₃): δ 7.68 (dd, 2H, J = 8.7, 2.7Hz), 7.38 (dd, 2H, J = 8.7, 4.2 Hz), 7.23 (td, 2H, J = 9.0, 2.7 Hz),4.50 (m, 1H), 4.39 (dd, 1H, J = 15.0, 4.8 Hz), 4.33 (dd, 1H, J = 15.0,7.8 Hz), 3.62-3.55 (m, 2H), 3.54- 3.35 (m, 2H), 2.75 (d, 1H, J = 4.5Hz), 2.40-2.20 (m, 2H), 2.08-1.96 (m, 2H). 395.1 (M + H) 14

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxy-2- methylpropyl)-3,3-difluoropiperidin-2- one (300 MHz, CDCl₃): δ 7.68 (dd, 2H, J = 8.7, 2.7Hz), 7.42 (dd, 2H, J = 8.7, 4.2 Hz), 7.22 (td, 2H, J = 9.0, 2.7 Hz),4.32 (s, 2H), 3.78 and 3.60 (ABq, 2H, J = 13.8 Hz), 3.73-3.58 (m, 2H),2.60 (s, 1H), 2.45-2.28 (m, 2H), 2.18-2.00 (m, 2H), 1.32 (s, 3H). 409.0(M + H) 15

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxypropyl)-3,3-dimethylpyrrolidin- 2-one (300 MHz, CDCl₃): δ 7.6S-7.65 (dd, 2H, J =2.7, 8.7 Hz), 7.38-7.33 (dd, 2H, J = 4.1, 9.0 Hz), 7.21-7.14 (td, 2H, J= 2.5, 9.0 Hz), 4.37-4.28 (m, 3H), 3.98-3.97 (d, 1H, J = 3.9 Hz),3.56-3.49 (dd, 1H, J = 7.1, 14.4 Hz), 3.34-3.17 (m, 3H), 1.90-1.85 (m,2H), 1.18 (s, 3H), 1.17 (s, 3H). 373.1 (M + H) 16

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxypropyl)-3,3dimethylpiperidin-2- one (300 MHz, CDCl₃): δ 7.67-7.64 (dd, 2H, J = 2.4,8.7 Hz), 7.38-7.34 (dd, 2H, J = 4.2, 8.7 Hz), 7.24-7.17 (td, 2H, J =2.5, 9.0 Hz), 4.54-4.53 (d, 1H, J = 3.0 Hz), 4.36-4.25 (m, 3H),3.94-3.86 (m, 1H), 3.18-3.11 (m, 2H), 2.95-2.90 (m, 1H), 1.81-1.61 (m,4H), 1.22 (s, 3H), 1.20 (s, 3H). 387.1 (M + H) 17

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2 hydroxypropyl)-3-ethylpyrrolidin-2- one (300 MHz, CDCl₃): δ 7.68-7.64 (dd, 2H, J = 2.7,8.7 Hz), 7.37-7.33 (dd, 2H, J = 4.1, 9.0 Hz), 7.21-7.14 (td, 2H, J =2.6, 9.0 Hz), 4.36-4.26 (m, 3H), 4.06-4.05 (d, 1H, J = 3.0 Hz),3.57-3.16 (m, 4H), 2.44-2.34 (m, 1H), 2.24-2.12 (m, 1H), 1.91-1.82 (m,1H), 1.77-1.66 (m, 1H), 1.49- 1.37 (m, 1H), 0.99-0.94 (td, 3H, J = 1.9,7.5 Hz). 373.1 (M + H) 18

3-cyclopentyl-1-(3- (3,6-difluoro-9H- carbazol-9-yl)-2- hydroxypropyl)pyrrolidin-2-one (300 MHz, CDCl₃): δ 7.63-7.64 (dd, 2H, J = 2.6, 8.6Hz), 7.37-7.33 (dd, 2H, J = 4.1, 8.9 Hz), 7.24-7.17 (td, 2H, J = 2.7,9.0 Hz), 4.36-4.26 (m, 3H), 4.09-4.00 (dd, 1H, J = 2.7, 24.9 Hz),3.59-3.43 (m, 1H), 3.35- 3.14 (m, 3H), 2.55-2.46 (m, 1H), 2.15-2.06 (m,2H), 1.94-1.63 (m, 7H), 1.52-1.19 (m, 2H). 413.1 (M + H) 19

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxypropyl)-3-phenylpiperidin-2- one (300 MHz, CDCl₃): δ 7.69-7.65 (dd, 2H, J = 2.7,8.7 Hz), 7.41-7.13 (m, 9H), 4.49-4.15 (m, 4H), 4.05-3.93 (m, 1H),3.71-3.63 (m, 1H), 3.37- 3.21 (m, 2H), 3.13-3.03 (m, 1H), 2.18-2.07 (m,1H), 1.99-1.70 (m, 3H). 435.1 (M + H) 20

3-cyclohexyl-1-(3- (3,6-difluoro-9H- carbazol-9-yl)-2- hydroxypropyl)piperidin-2-one (300 MHz, CDCl₃, mixture of diastereomers): δ 7.67-7.66(two overlapping sets of dd, 2H, J = 2.3, 8.7 Hz), 7.39-7.34 (twooverlapping sets of dd, 2H, J = 4.2, 8.7 Hz), 7.24- 7.16 (twooverlapping sets of td, 2H, J = 2.6, 9.0 Hz), 4.90 (s, 0.5H), 4.40- 4.23(m, 3.5H), 4.16-4.15 (d, 0.5H, J = 3.0 Hz), 3.95-3.87 (m, 1H), 3.28-3.21 (m, 0.5H), 3.14-3.02 (m, 2.5H), 2.89-2.84 (dd, 0.5H, J = 1.5, 14.1Hz), 2.29-2.08 (m, 2H), 1.84-0.99, (m, 14H). 441.2 (M + H) 21

3-cyclohexyl-1-(3- (3,6-difluoro-9H- carbazol-9-yl)-2- hydroxy-2-methylpropyl) piperidin-2-one (300 MHz, CDCl₃, mixture ofdiastereomers): δ 7.66-7.62 (two overlapping sets of dd, 2H, J = 2.4,8.7 Hz), 7.45-7.41 (dd, 2H, J = 3.9, 9.0 Hz), 7.22-7.15 (two sets ofoverlapping td, 2H, J = 2.4, 9.0 Hz), 5.24 (s, 0.5H), 5.11 (s, 0.5 H),4.31- 4.07 (m, 2H), 4.12-4.07, 3.12-3.07 (ABq, 1H, J = 14.1 Hz),3.92-3.87, 3.25-3.16 (ABq, 1H, J = 14.1 Hz), 3.55-3.25 (m, 2H),2.37-2.05 (m, 2H), 1.93-0.84 (m, 14H), 1.24 (s, 3H). 455.1 (M + H) 22

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxypropyl)-3-isopropylpyrrolidin- 2-one (300 MHz, CDCl₃, mixture of diastereomers): δ7.68-7.64 (dd, 2H, J = 2.4, 8.7 Hz), 7.37-7.33 (dd, 2H, J = 4.0, 9.0Hz), 4.36-4.26 (m, 3H), 4.10-4.00 (m, 1H), 3.61-3.14 (m, 4H), 2.49-2.40(m, 1H), 2.24-2.17 (m, 1H), 2.04-1.98 (m, 1H), 1.89- 1.79 (m, 1H),1.01-0.98 (two overlapping sets of d, 3H, J = 6.9 Hz), 0.89-0.86 (twooverlapping sets of d, 3H, J = 6.9 Hz). 387.1 (M + H) 23

3-cyclopentyl-1-(3- (3,6-difluoro-9H- carbazol-9-yl)-2- hydroxypropyl)piperidin-2-one (300 MHz, CDCl₃, mixture of diastereomers): δ 7.68-7.64(dd, 1H, J = 2.1, 8.4 Hz), 7.67-7.63 (dd, 1H, J = 2.3, 9.0 Hz),7.39-7.34 (dd, 2H, J = 4.4, 9.0 Hz), 7.24-7.17 (td, 1H, J = 2.6, 9.0Hz), 7.23-7.17 (td, 1H, J = 2.4, 9.0 Hz), 4.76-4.75 (d, 0.5H, J = 2.4Hz), 4.38-4.23 (m, 3.5H), 3.98-3.84 (m, 1H), 3.25-2.90 (m, 3H),2.41-2.29 (m, 1H), 1.87-1.15 (m, 12H). 427.1 (M + H) 24

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxypropyl)-3-ethylpiperidin-2-one (300 MHz, CDCl₃, mixture of diastereomers): δ7.67-7.64 (dd, 1H, J = 2.4, 8.7 Hz), 7.67-7.64 (dd, 1H, J = 2.4, 8.7Hz), 7.38-7.34 (dd, 2H, J = 4.2, 9.0 Hz), 7.24-7.17 (td, 1H, J = 2.7,9.0 Hz), 7.24-7.16 (td, 1H, J = 2.7, 9.0 Hz), 4.70 (br s, 0.5H),4.38-4.26 (m, 3.5H), 3.95-3.83 (m, 1H), 3.20-2.87 (m, 3H), 2.28-2.20 (m,1H), 2.00-1.43 (m, 6H), 0.96- 0.89 (m, 3H). 387.1 (M + H) 25

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxypropyl)-3-isopropylpiperidin- 2-one (300 MHz, CDCl₃, mixture of diastereomers): δ7.67-7.64 (dd, 1H, J = 2.1, 8.7 Hz), 7.67-7.63 (dd,1H, J = 2.4, 8.7 Hz),7.38-7.34 (dd, 2H, J = 4.2, 8.7 Hz), 7.24-7.17 (td, 1H, J = 2.4, 9.0Hz), 7.24-7.16 (td, 1H, J = 2.4, 9.0 Hz), 4.83 (br s, 0.5H), 4.38-4.22(m, 3H), 4.15 (br s, 0.5H), 3.95-3.88 (m, 1H), 3.31-3.22 (m, 0.5H),3.16-2.86 (m, 2.5H), 2.59- 2.47 (m, 1H), 2.32-2.20 (m, 1H), 1.87-1.39(m, 4H), 0.96-0.93 (dd, 401.1 (M + H) 3H, J = 2.0, 7.2 Hz), 0.85-0.77(dd, 3H, J = 6.6, 15.6 Hz). 26

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxy-2- methylpropyl)-3-isopropylpiperidin- 2-one (300 MHz, CDCl₃, mixture of diastereomers): δ7.66-7.62 (dd, 2H, J = 2.6, 8.7 Hz), 7.45-7.41 (two overlapping sets ofdd, 2H, J = 4.1, 8.7 Hz), 7.21-7.14 (td, 2H, J = 2.7, 9.0 Hz), 5.14 (s,0.5H), 5.03 (s, 0.5 H), 4.31-4.09 (m, 2.5 H), 3.92-3.87, 3.21- 3.17(ABq, 1H, J = 14.1 Hz), 3.57-3.25 (m, 2H), 3.11-3.06 (d, 0.5H, J = 14.1Hz), 2.59-2.50 (m, 1H), 2.38-2.26 (m, 1H), 1.95-1.49 (m, 5H), 1.27 (s,3H), 0.97-0.94 (dd, 415.1 (M + H) 3H, J = 2.0, 7.5 Hz), 0.87-0.80 (dd,3H, J = 6.9, 14.4 Hz). 27

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxypropyl)-3-methylpiperidin-2- one (300 MHz, CDCl₃, mixture of diastereomers): δ7.68-7.64 (dd, 2H, J = 2.6, 8.7 Hz), 7.39-7.35 (dd, 2H, J = 4.2, 8.7Hz), 7.25-7.17 (td, 2H, J = 2.4, 9.0 Hz), 4.60-4.59 (d, 0.5H, J = 3.0Hz), 4.39-4.27 (m, 3.5H), 3.97- 3.80 (m, 1H), 3.25-3.04 (m, 2.5H),2.91-2.86 (m, 0.5H), 2.46-2.39 (m, 1H), 1.99-1.62 (m, 3H), 1.55-1.39 (m,1H), 1.26-1.21 (t, 3H, J = 7.5 Hz). 373.1 (M + H) 28

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxy-2- methylpropyl)-3-methylpiperidin-2- one (300 MHz, CDCl₃, mixture of diastereomers): δ7.65-7.62 (dd, 2H, J = 2.6, 8.6 Hz), 7.45-7.40 (dd, 2H, J = 4.2, 9.2Hz), 7.21-7.14 (td, 2H, J = 2.7, 9.0 Hz), 5.02 (s, 0.5H), 4.88 (s,0.5H), 4.25 (s, 2H), 3.98-3.89 (t, 1H, J = 14.1 Hz), 3.53-3.30 (m, 2H),3.21-3.17 (d, 0.5H, J = 14.1 Hz), 3.10-3.05 (d, 0.5H, J = 14.1 Hz),2.51-2.43 (m, 1H), 2.03-1.75 (m, 3H), 1.67-1.46 (m, 1H), 1.28 (s, 1.5H),1.27-1.25 (d, 1.5H, J = 7.5 Hz), 387.0 (M + H) 1.26 (s, 1.5H), 1.23-1.21(d, 1.5H, J = 7.5 Hz). 29

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxypropyl)-3-methylpyrrolidin-2- one (300 MHz, CDCl₃, mixture of diastereomers): δ7.69-7.65 (dd, 2H, J = 2.6, 8.7 Hz), 7.38-7.33 (dd, 2H, J = 4.1, 9.0Hz), 4.38-4.28 (m, 3H), 4.00-3.94 (dd, 1H, J = 4.2, 14.7 Hz), 3.56-3.15(m, 4H), 2.55-2.50 (m, 1H), 2.28-2.21 (m, 1H), 1.69-1.60 (m, 1H),1.23-1.21 (d, 3H, J = 6.9 Hz). 359.1 (M + H) 30

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxy-2- methylpropyl)-3-methylpyrrolidin-2- one (300 MHz, CDCl₃): δ 7.65-7.61 (dd, 2H, J = 2.7,8.7 Hz), 7.43-7.38 (dd, 2H, J = 4.4, 8.9 Hz), 7.20-7.13 (td, 2H, J =2.6, 8.9 Hz), 4.30-4.20 (m, 2H), 3.75-3.73 (d, 1H, J = 3.3 Hz),3.59-3.27 (m, 4H), 2.54-2.48 (m, 1H), 2.30-2.26 (m, 1H), 1.75-1.65 (m.1H), 1.29-1.18 (m, 6H). 373.0 (M + H) 31

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxypropyl)-4,4-dimethylpyrrolidin- 2-one (300 MHz, CDCl₃): δ 7.68-7.65 (dd, 2H, J =2.6, 8.6 Hz), 7.38-7.33 (dd, 2H, J = 4.1, 9.1 Hz), 7.25-7.17 (td, 2H, J= 2.5, 9.1 Hz), 4.37-4.33 (m, 3H), 3.62-3.52 (m, 2H), 3.26-3.21 (m, 1H),3.17-3.14, 3.12-3.08 (ABq, 2H, J = 9.4 Hz), 2.25 (s, 2H), 1.16 (s, 2H),1.16 (s, 3H), 1.14 (s, 1H). 373.1 (M + H) 32

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxypropyl)-5-ethylpyrrolidin-2- one (300 MHz, CDCl₃, unequal mixture of diastereomersapprox. 1:4): δ 7.69-7.65 (dd, 2H, J = 2.6, 8.6 Hz), 7.38-7.33 (dd, 2H,J = 4.1, 8.9 Hz), 7.25-7.18 (td, 2H, J = 2.5, 9.0 Hz), 4.80-4.79 (d,0.2H, J = 2.7 Hz), 4.39-4.26 (m, 3H), 4.09-4.08 (m, 0.8H), 3.72-3.64 (m,1H), 3.48-3.40 (m, 1H), 3.12-3.03 (m, 1H), 2.44- 2.33 (m, 2H), 2.22-2.09(m, 1H), 1.76-1.66 (m, 1H), 1.57-1.49 (m, 373.0 (M + H) 1H), 1.28-1.18(m, 1H), 0.81-0.76 (t, 2.5H, J = 7.5 Hz), 0.65-0.60 (t, 0.5H, J = 7.4Hz). 33

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxy-2- methylpropyl)-5-ethylpyrrolidin-2- one (300 MHz, d⁶-DMSO): δ 7.66-7.62 (dd, 2H, J = 2.3Hz, 8.9 Hz), 7.46- 7.41 (dd, 2H, J = 4.1, 8.9 Hz), 7.22- 7.15 (td, 2H, J= 2.6, 8.9 Hz), 4.33- 4.19 (m, 3H), 3.67-3.57 (m, 2H), 3.28-3.23 (d, 1H,J = 14.4 Hz), 2.94 (s, 0.5H), 2.86-2.86 (d, 0.5H, J = 3.3Hz), 2.50-2.32(m, 2H), 2.24-2.12 (m, 1H), 1.80-1.70 (m, 2H), 1.14- 1.34 (m, 1H), 1.22(s, 3H), 0.91-0.86 (t, 3H, J = 7.4 Hz). 387.0 (M + H) 34

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxypropyl)-5-methylpyrrolidin-2- one (300 MHz, CDCl₃): δ 7.68-7.64 (dd, 2H, J = 2.6,8.6 Hz), 7.38-7.33 (dd, 2H, J = 4.1, 8.9 Hz), 7.24-7.18 (m, 2H),4.73-4.72 (d, 0.5H, J = 3.0 Hz), 4.38-4.25 (m, 3H), 4.08-4.06 (d, 0.5H,J = 3.6 Hz), 3.69-3.57 (m, 1H), 3.49-3.35 (m, 1H), 3.11-3.04 (m, 1H),2.49-2.01 (m, 3H), 1.67- 1.53 (m, 1H), 1.05-1.03 (d, 1.5H, J = 6.3 Hz),0.84-0.82 (d, 1.5H, J = 6.3 Hz). 359.1 (M + H) 35

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxy-2- methylpropyl)-5-methylpyrrolidin-2- one (300 MHz, CDCl₃): δ 7.67-7.63 (dd, 2H, J = 2.6,8.9 Hz), 7.46-7.42 (dd, 2H, J = 4.1, 8.9 Hz), 7.23-7.16 (td, 2H, J =2.7, 9.0 Hz), 4.34-4.21 (m, 3H), 3.82-3.72 (sext, 1H, J = 6.6 Hz),3.60-3.56, 3.32-3.27 (ABq, 2H, J = 14.4 Hz), 2.49-2.20 (m, 3H),1.71-1.61 (m, 1H), 1.24 (s, 3H), 1.23-1.21 (d, 3H, J = 6.6 Hz). 373.0(M + H) 36

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxy-2- methylpropyl)-4-methylpiperidin-2- one (300 MHz, CDCl₃): δ 7.67-7.63 (dd, 2H, J = 2.6,9.0 Hz), 7.46-7.41 (dd, 2H, J = 4.1, 8.9 Hz), 7.22-7.15 (td, 2H, J =2.5, 8.9 Hz), 4.96 (s, 0.5H), 4.72 (s, 0.5H), 4.28 (s, 2H), 4.05- 4.01,3.34-3.29 (ABq, 1H, J = 14.1 Hz), 3.89-3.84, 3.06-3.01 (ABq, 1H, J =14.1 Hz), 3.57-3.37 (m, 2H), 2.60-2.48 (m, 1H), 2.10-1.84 (m, 2H),1.57-1.48 (m, 2H), 1.28 (s, 3H), 1.05-1.03 (d, 3H, J = 6.3 Hz); HPLCanalysis: (C18, 10-90% acetonitrile 387.1 (M + H) in water over 20 min:retention time, % area at 254 nm): 13.2 min, 98%. 37

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxypropyl)-5-methylpiperidin-2- one (300 MHz, CDCl₃): δ 7.67-7.62 (m, 2H), 7.38-7.33(dd, 2H, J = 4.1, 9.0 Hz), 7.23-7.16 (m, 2H), 4.55-4.54 (d, 0.5H, J =3.3 Hz), 4.37-4.24 (m, 3H), 4.20-4.18 (d, 0.5H, J = 3.6 Hz), 3.88-3.81(dd, 0.5H, J = 8.6, 14.1 Hz), 3.77-3.70 (dd, 0.5H, J = 8.4, 14.1 Hz),3.18-2.76 (m, 2H), 2.49- 2.28 (m, 2H), 1.92-1.77 (m, 2H), 1.49-1.30 (m,1H), 0.94-0.93 (d, 1.5H, J = 3.0 Hz), 0.92-0.91 (d, 1.5H, J = 3.0 Hz).373.1 (M + H) 38

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxy-2- methylpropyl)-5-methylpiperidin-2- one (300 MHz, CDCl₃): δ 7.67-7.63 (dd, 2H, J = 2.4,8.9 Hz), 7.46-7.41 (dd, 2H, J = 4.1, 8.9 Hz), 7.22-7.15 (td, 2H, J =2.4, 9.0 Hz), 4.97 (s, 0.5H), 4.76 (s, 0.5H), 4.33-4.22 (m, 2H), 4.02(d, 0.5H, J = 14.1 Hz), 3.90- 3.85 (d, 0.5H, J = 14.1 Hz), 3.50- 2.95(m, 3H), 2.57-2.38 (m, 2H), 2.01-1.85 (m, 2H), 1.62-1.40 (m, 1H), 1.29(s, 3H), 1.04-0.99 (t, 3H, J = 6.8 Hz). 387.1 (M + H) 39

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxypropyl)-6-methylpiperidin-2- one (300 MHz, CDCl₃): δ 7.68-7.64 (dd, 2H, J = 2.6,8.6 Hz), 7.40-7.35 (dd, 2H, J = 4.2, 9.0 Hz), 7.25-7.18 (td, 1H, J =2.7, 9.0 Hz), 7.25-7.18 (td, 1H, J = 2.5, 9.0 Hz), 5.51-5.50 (d, 0.5H, J= 2.1 Hz), 4.84-4.83 (d, 0.5H, J = 2.4 Hz), 4.39-4.22 (m, 3H), 4.06-3.98(dd, 0.5H, J = 8.6, 14.4 Hz), 3.83-3.76 (dd, 0.5H, J = 8.0, 14.1 Hz),3.21-3.13 (m, 1H), 2.96-2.91 (dd, 0.5H, J = 0.9, 14.1 Hz), 2.86-2.81(dd, 0.5H, J = 1.5, 373.1 (M + H) 14.1 Hz), 2.41-2.35 (m, 2H), 1.87-1.38 (m, 4H), 0.96-0.94 (d, 1.5H, J = 6.6 Hz), 0.67-0.65 (d, 1.5H, J =6.6 Hz). 40

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxy-2- methylpropyl)-6-methylpiperidin-2- one (300 MHz, CDCl₃): δ 7.66-7.62 (dd, 2H, J = 2.6,9.0 Hz), 7.45-7.41 (dd, 2H, J = 4.2, 9.0 Hz), 7.21-7.15 (td, 2H, J =2.4, 9.0 Hz), 5.13 (s, 1H), 4.34-4.29, 4.21-4.16 (ABq, 2H, J = 14.9 Hz),4.05-4.00 (d, 1H, J = 14.7 Hz), 3.68-3.64 (m, 1H), 3.26-3.21 (d, 1H, J =14.4 Hz), 2.52-2.47 (m, 2H), 1.96-1.70 (m, 4H), 1.26-1.23 (d, 3H, J =6.6 Hz), 1.22 (s, 3H). 373.1 (M + H) 41

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxypropyl)-4-isopropylpyrrolidin- 2-one (300 MHz, CDCl₃): δ 7.69-7.65 (dd, 2H, J =2.6, 8.6 Hz), 7.38-7.33 (dd, 2H, J = 4.1, 8.9 Hz), 7.25-7.18 (m, 2H),4.37-4.28 (m, 3H), 3.87-3.85 (m, 1H), 3.58-3.03 (m, 5H), 2.55- 2.46 (m,1H), 2.21-2.02 (m, 2H), 0.92-0.89 (dd, 3H, J = 3.2, 6.8 Hz), 0.86-0.84(dd, 3H, J = 1.5, 6.3 Hz). 387.1 (M + H) 42

4-cyclopropyl-1-(3- (3,6-difluoro-9H- carbazol-9-yl)-2- hydroxypropyl)pyrrolidin-2-one (300 MHz, CDCl₃): δ 7.69-7.65 (dd, 2H, J = 2.4, 8.4Hz), 7.38-7.34 (dd, 2H, J = 4.1, 8.9 Hz), 7.25-7.18 (td, 2H, J = 2.4,9.0 Hz), 4.40-4.29 (m, 3H), 3.80-3.79 (d, 1H, J = 3.9 Hz), 3.60-3.17 (m,4H), 2.61-2.52 (dd, 1H, J = 8.9, 17.0 Hz), 2.33-2.23 (m, 1H), 1.76-1.63(m, 1H), 0.80-0.75 (m, 1H), 0.56-0.45 (m, 2H), 0.19- 0.08 (m, 2H). 385.1(M + H) 43

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxypropyl)-4-methylpyrrolidin-2- one (300 MHz, CDCl₃): δ 7.69-7.65 (dd, 2H, J = 2.6,8.6 Hz), 7.38-7.33 (dd, 2H, J = 4.1, 8.9 Hz), 7.25-7.18 (td, 2H, J =2.6, 8.9 Hz), 4.37-4.28 (m, 3H), 3.77-3.75 (m, 1H), 3.57-3.38 (m, 2H),3.28-3.20 (m, 1H), 3.03- 2.93 (m, 1H), 2.64-2.55 (dd, 1H, J = 8.6, 16.5Hz), 2.53-2.40 (dd, 1H, J = 6.9 Hz), 2.12-2,08 (t, 0.5H, J = 6.6 Hz),2.06-2.02 (t, 0.5H, J = 2.5 Hz), 1.13-1.10 (d, 1.5H, J = 6.6 Hz), 359.1(M + H) 1.16-1.09 (d, 1.5H, J = 6.6 Hz). 44

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxy-2- methylpropyl)-4-methylpyrrolidin-2- one (300 MHz, CDCl₃): δ 7.67-7.64 (dd, 2H, J = 2.4,8.7 Hz), 7.44-7.40 (dd, 2H, J = 3.8, 8.6 Hz), 7.22-7.16 (td, 2H, J =2.5, 9.0 Hz), 4.34-4.23 (m, 2H), 3.74-3.50 (m, 3H), 3.38-3.32 (dd, 1H, J= 3.6, 14.1 Hz), 3.24-3.19 (dd, 0.5H, J = 5.4, 10.2 Hz), 3.16- 3.11 (dd,0.5H, J = 6.5, 6.6 Hz), 2.66-2.46 (m, 2H), 2.14-2.05 (m, 1H), 1.27 (s,3H), 1.19-1.17 (d, 1.5H, J = 6.3 Hz), 1.16-1.14 (d, 373.0 (M + H) 1.5H,J = 6.3 Hz). 45

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxypropyl)-4-ethylpyrrolidin-2- one (300 MHz, CDCl₃): δ 7.69-7.65 (dd, 2H, J = 2.6,8.6 Hz), 7.38-7.33 (dd, 2H, J = 4.1, 9.0 Hz), 7.24-7.18 (td, 2H, J =2.6, 9.0 Hz), 4.37-4.27 (m, 3H), 3.81 (d, 1H, J = 2.1 Hz), 3.58- 3.19(m, 3H), 3.08-2.98 (m, 1H), 2.60-2.52 (dd, 1H, J = 8.7, 16.2 Hz),2.31-2.23 (m, 1H), 2.16-2.01 (m, 1H), 1.50-1.39 (m, 1H), 0.93-0.88 (t,1.5H, J = 7.5 Hz), 0.92-0.97 (t, 1.5H, J = 7.5 Hz). 373.1 (M + H) 46

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxy-2- methylpropyl)-4-ethylpyrrolidin-2- one (300 MHz, CDCl₃): δ 7.67-7.63 (dd, 2H, J = 2.3,8.9 Hz), 7.44-7.40 (dd, 2H, J = 4.1, 8.9 Hz), 7.22-7.16 (td, 2H, J =2.4, 9.0 Hz), 4.33-4.22 (m, 2H), 3.71-3.53 (m, 3H), 3.39-3.15 (m, 2H),2.63-2.54 (ddd, 1H, J = 2.7, 8.4, 16.8 Hz), 2.38-2.27 (sept, 1H, J = 7.8Hz), 2.19-2.11 (m, 1H), 1.54- 1.44 (m, 2H), 1.26 (s, 3H), 0.98-0.91 (m,3H). 387.1 (M + H) 47

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxypropyl)-4,5-dimethylpyrrolidin- 2-one (300 MHz, CDCl₃): δ 7.68-7.64 (dd, 2H, J =2.6, 8.6 Hz), 7.37-7.33 (dd, 2H, J = 4.1, 8.9 Hz), 7.24-7.17 (td, 2H, J= 2.5, 9.0 Hz), 4.64-4.59 (m, 0.5H), 4.38-4.24 (m, 3.5H), 3.88- 2.97 (m,3H), 2.63-2.40 (m, 2H), 2.13-1.88 (m, 1H), 1.11-0.77 (m, 6H). 373.1 (M +H) 48

2-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxypropyl)-2-azabicyclo[2.2.1] heptan-3-one (300 MHz, CDCl₃): δ 7.69-7.66 (dd, 1H, J= 2.1, 8.7 Hz), 7.69-7.65 (dd, 1H, J = 2.1, 8.7 Hz), 7.38-7.34 (dd, 1H,J = 3.9, 8.7 Hz), 7.38-7.33 (dd, 1H, J = 2.1, 8.7 Hz), 7.25-7.18 (td,1H, J = 3.0, 9.0 Hz), 7.24-7.17 (td, 1H, J = 3.0, 9.0 Hz), 4.40-4.25 (m,4H), 3.73 (brs, 0.5H), 3.64 (br s, 0.5H), 3.48-3.39 (m, 1H), 3.03-2.85(m, 2H), 1.96-1.55 (m, 5H), 1.43- 1.36 (m, 1H). 371.1 (M + H) 49

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxypropyl)-4-methylpiperidin-2- one (300 MHz, CDCl₃): δ 7.68-7.64 (dd, 2H, J = 2.7,8.7 Hz), 7.39-7.35 (dd, 2H, J = 4.2, 8.7 Hz), 7.24-7.17 (td, 1H, J =2.6, 9.0 Hz), 7.23-7.17 (td, 1H, J = 2.6, 9.0 Hz), 4.52 (br s, 0.5H),4.39-4.27 (m, 3H), 4.20-4.19 (d, 0.5H, J = 3.0 Hz), 3.97-3.89 (m, 0.5H),3.85-3.78 (dd, 0.5H, J = 8.0, 14.1 Hz), 3.28-3.07 (m, 2.5H), 2.94- 2.89(m, 0.5H), 2.53-2.45 (m, 1H), 2.05-1.75 (m, 3H), 1.48-1.33 (m, 1H),1.01-0.98 (d, 3H, J = 6.3 Hz). 373.1 (M + H) 50

3-cyclobutyl-1-(3- (3,6-difluoro-9H- carbazol-9-yl)-2- hydroxypropyl)pyrrolidin-2-one (300 MHz, CDCl₃): δ 7.68-7.64 (dd, 2H, J = 2.6, 8.6Hz), 7.37-7.33 (dd, 2H, J = 14.1, 8.9 Hz), 7.24-7.17 (td, 2H, J = 2.5,9.0 Hz), 4.36-4.27 (m, 3H), 4.05-4.03 (d, 0.5H, J = 4.5 Hz), 3.95-3.94(d. 0.5H, J = 3.9 Hz), 3.55-3.44 (m, 1H), 3.31-3.16 (m, 3H), 2.55-2.48(m, 2H), 2.15-2.73 (m, 8H). 399.1 (M + H) 51

3-cyclobutyl-1-(3- (3,6-difluoro-9H- carbazol-9-yl)-2- hydroxypropyl)piperidin-2-one (300 MHz, CDCl₃): δ 7.68-7.64 (dd, 2H, J = 2.6, 8.6 Hz),7.39-7.34 (dd, 1H, J = 4.1, 8.7 Hz), 7.38-7.34 (dd, 1H, J = 4.2, 9.0Hz), 7.24-7.17 (td, 2H, J = 2.6, 8.9 Hz), 4.60-4.59 (d, 0.5H, J = 3.3Hz), 4.38-4.25 (m, 3H), 4.24-4.22 (d, 0.5H, J = 3.6 Hz), 3.95-3.84 (m,1H), 3.17-2.87 (m, 3H), 2.57-2.51 (m, 1H), 2.34-2.26 (m, 1H), 2.14-1.39(m, 9H). 413.1 (M + H) 52

3-cyclobutyl-1-(3- (3,6-difluoro-9H- carbazol-9-yl)-2- hydroxy-2-methylpropyl) piperidin-2-one (300 MHz, CDCl₃): δ 7.66-7.62 (dd, 2H, J =2.6, 8.9 Hz), 7.45-7.41 (dd, 2H, J = 4.1, 8.9 Hz), 7.21-7.15 (td, 1H, J= 2.6, 8.9 Hz), 7.21-7.14 (td, 1H, J = 2.6, 8.9 Hz), 4.92 (s, 0.5H),4.87 (s, 0.5H), 4.31-4.20 (m, 2H), 3.22-3.18 (ABq, 1H, J = 14.1 Hz),3.88-3.83, 3.06-3.02 (ABq, 1H, J = 14.1 Hz), 3.49-3.27 (m, 2H), 2.63-2.52 (m, 1H), 2.39-2.27 (m, 1H), 2.16-1.47 (m, 10H), 1.27 (s, 3H). 413.1(M + H) 53

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxypropyl)-3-phenylpyrrolidin-2- one (300 MHz, CDCl₃): δ 7.69-7.65 (dd, 2H, J = 2.6,8.6 Hz), 7.37-7.17 (m, 9H), 4.40-4.32 (m, 3H), 3.80-3.26 (m, 6H),2.57-2.46 (m, 1H), 2.23- 2.11 (m, 1H). 421.1 (M + H) 54

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxy-2- methylpropyl)-3-phenylpyrrolidin-2- one (300 MHz, CDCl₃): δ 7.67-7.64 (dd, 2H, J = 2.4,8.7 Hz), 7.44-7.40 (dd, 2H, J = 3.9, 9.0 Hz), 7.36-7.15 (m, 7H),4.37-4.25 (m, 2H), 3.79-3.31 (m, 6H), 2.62-2.54 (m, 1H), 2.29- 2.16 (m,1H), 1.31 (s, 1.5H), 1.30 (s, 1.5H). 435.1 (M + H) 55

4-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxypropyl) morpholin-3-one(300 MHz, CDCl₃): δ 7.68-7.64 (dd, 2H, J = 2.6, 8.6 Hz), 7.39-7.34 (dd,2H, J = 4.1, 9.0 Hz), 7.24-7.18 (td, 2H, J = 2.4, 9.0 Hz), 4.43-4.41 (m,1H), 4.34 (s, 1H), 4.32 (d, 1H, J = 0.9 Hz), 4.18 (s, 2H), 3.84-3.79 (m,2H), 3.76-3.68 (dd, 1H, J = 8.6, 17.4 Hz), 3.53-3.51 (d, 1H, J = 3.9Hz), 3.41-3.28 (m, 3H). 361.1 (M + H) 56

4-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxy-2- methylpropyl)morpholin-3-one (300 MHz, CDCl₃): δ 7.67-7.64 (dd, 2H, J = 2.4, 8.7 Hz),7.44-7.40 (dd, 2H, J = 4.1, 8.9 Hz), 7.23-7.16 (td, 1H, J = 2.4, 8.9Hz), 4.30 (s, 2H), 4.24 (s, 2H), 3.91-3.86 (m, 3H), 3.61-3.56 (m, 2H),3.53 (s, 1H), 3.40-3.35 (d, 1H, J = 14.1 Hz), 1.31 (s, 3H). 375.0 (M +H) 57

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxypropyl)-3-methoxypiperidin-2- one (300 MHz, CDCl₃): δ 7.67-7.63 (dd, 2H, J = 2.3,8.9 Hz), 7.39-7.34 (dd, 2H, J = 4.2, 8.7 Hz), 7.24-7.17 (td, 1H, J =2.6, 9.0 Hz), 7.23-7.16 (td, 1H, J = 2.6, 9.0 Hz), 4.38-4.30 (m, 3H),3.90-3.59 (m, 3H), 3.53 (s, 3H), 3.32-3.06 (m, 3H), 1.99-1.88 (m, 2H),1.70-1.65 (m, 2H). 389.1 (M + H) 58

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxypropyl)-3,3-difluoropyrrolidin-2- one (300 MHz, CDCl₃): δ 7.69-7.65 (dd, 2H, J =2.4, 8.7 Hz), 7.37-7.32 (dd, 2H, J = 3.9, 8.7 Hz), 7.25-7.18 (td, 2H, J= 3.0, 8.7 Hz), 4.49-4.43 (m, 1H), 4.36-4.32 (m, 2H), 3.62-3.45 (m, 4H),2.67-2.66 (d, 1H, J = 4.5 Hz), 2.67-2.45 (m, 2H); ¹⁹F NMR (282 MHz,CDCl₃): δ −105.1 to −105.2 (m), −123.57 to −123.4 (t, J = 10.7 Hz). NA59

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxy-2- methylpropyl)-3,3-difluoropyrrolidin-2- one (300 MHz, CDCl₃): δ 7.68-7.64 (dd, 2H, J =2.7, 8.6 Hz), 7.41-7.36 (dd, 2H, J = 4.1, 8.9 Hz), 7.24-7.17 (td, 2H, J= 2.7, 8.7 Hz), 4.30 (s, 2H), 3.74-3.69 (m, 2H), 3.67-3.62, 3.56- 3.51(ABq, 2H, J = 14.1 Hz), 2.64- 2.49 (m, 2H), 2.14 (s, 1H), 1.30 (s, 3H);¹⁹F NMR (282 MHz, CDCl₃): δ −105.6 to −105.74 (t, J = 15.2 Hz), −123.6to −123.6 (t, J = 10.6 Hz). NA 60

1-(3-(9H-carbazol- 9-yl)-2- hydroxypropyl)-3- methoxypiperidin-2- one(300 MHz, CDCl₃): δ 8.07 (d, 2H, J = 6.9 Hz), 7.47-7.41 (m, 4H), 7.27-7.19 (m, 2H), 4.41-4.26 (m, 3H), 3.96 (dd, 1H, J = 6.6, 11.1 Hz), 3.76-3.52 (m, 2H), 3.50 (s, 3H), 3.29-2.98 (m, 3H), 1.97-1.78 (m, 3H), 1.65-1.55 (m, 1H); HPLC analysis: (C18, 10-90% acetonitrile in water + 0.1%trifluoroacetic acid over 20 min: 354.1 (M + H) retention time, % areaat 254 nm): 11.2 min, 99.6%. 61

1-(3-(9H-carbazol- 9-yl)-2- hydroxypropyl)-4- methylpiperidin-2- one(300 MHz, CDCl₃): δ 8.09 (dd, 2H, J = 1.2, 7.2 Hz), 7.50-7.44 (m, 4H),7.29-7.20 (m, 2H), 4.54-4.14 (m, 4H), 3.98-3.81 (m, 1H), 3.20-2.64 (m,3H), 2.50-2.28 (m, 2H), 1.85- 1.74 (m, 2H), 1.48-1.28 (m, 1H), 0.88,0.87 (two overlapping dd, 3H, J = 6.6 Hz); HPLC analysis: (C18, 10-90%acetonitrile in water + 0.1% 337.7 (M + H) trifluoroacetic acid over 20min: retention time, % area at 254 nm): 12.1 min, 98.7%. 62

1-(3-(9H-carbazol- 9-yl)-2- hydroxypropyl)-5- methylpiperidin-2- one(300 MHz, CDCl₃): δ 8.09 (d, 2H, J = 7.2 Hz), 7.49-7.41 (m, 4H), 7.28-7.20 (m, 2H), 4.52 (br s, 0.5H), 4.44- 4.29 (m, 3H), 4.21 (d, 0.5H, J =3.6 Hz), 3.99-3.81 (m, 1H), 3.20-2.89 (m, 3H), 2.51-2.43 (m, 1H), 2.03-1.31 (m, 4H), 0.97 (d, 3H, J = 6.6 Hz); HPLC analysis: (C18, 10-90%acetonitrile in water + 0.1% 337.9 (M + H) trifluoroacetic acid over 20min: retention time, % area at 254 nm): 12.1 min, 98.5%. 63

1-(3-(9H-carbazol- 9-yl)-2- hydroxypropyl)-6- methylpiperidin-2- one(300 MHz, CDCl₃): δ 8.09 (d, 2H, J = 7.5 Hz), 2.48-2.45 (m, 4H), 7.27-7.21 (m, 2H), 5.50 (s, 0.6 H), 4.84 (d, 0.4H, J = 3.6 Hz), 4.46-4.27 (m,3 H), 4.07 (dd, 0.4 H, J = 8.1, 14.1 Hz), 3.82 (dd, 0.6H, J = 7.6, 14.0Hz), 3.15-3.07 (m, 1H), 2.97 (d, 0.6H, J = 14.1 Hz), 2.85 (d, 0.4H, J =14.1 Hz), 2.40-2.33 (m, 2H), 1.76- 337.5 (M + H) 1.32 (m, 4H), 0.86 (d,1.2H, J = 6.3 Hz), 0.59 (d, 1.8 H, J = 6.6 Hz); HPLC analysis: (C18,10-90% acetonitrile in water + 0.1% trifluoroacetic acid over 20 min:retention time, % area at 254 nm): 12.1 min, 95.2%. 64

1-(3-(9H-carbazol- 9-yl)-2- hydroxypropyl)-3- methylpiperidin-2- one(300 MHz, CDCl₃): δ 8.08 (d, 2H, J = 7.5 Hz), 7.45-7.42 (m, 4H), 7.28-7.21 (m, 2H), 4.59 (br s, 0.4H), 4.43- 4.26 (m, 3.6H), 3.94 (dd, 0.5H, J= 8.0, 13.7 Hz), 3.81 (dd, 0.5H, J = 7.5, 14.1 Hz), 3.17-2.89 (m, 3H),2.42-2.33 (m, 1H), 1.94-1.33 (m, 4H), 1.23-1.19 (m, 3H); HPLC analysis:(C18, 10-90% acetonitrile 337.9 (M + H) in water + 0.1% trifluoroaceticacid over 20 min: retention time, % area at 254 nm): 12.2 min, 96.5%. 65

1-(3-(9H-carbazol- 9-yl)-2- hydroxypropyl)-4- cyclopropyl-pyrrolidin-2-one (300 MHz, CDCl₃): δ 8.08 (d, 2H, J = 7.5 Hz), 7.48-7.42(m, 4H), 7.28- 7.21, m, 2H), 4.39-4.29 (m, 3H), 3.87 (d, 1H, J = 14.7Hz), 3.57-3.10 (m, 4H), 2.52 (dd, 0.5H, J = 5.1, 9.0 Hz), 2.46 (dd,0.5H, J = 8.3, 9.0 Hz), 2.24 (t, 0.5H, J = 6.9 Hz), 2.18 (t, 0.5H, J =6.8 Hz), 1.75-1.60 (m, 1H), 0.75-0.68 (m, 1H), 0.49-0.38 349.3 (M + H)(m, 2H), 0.14-0.02 (m, 2H); HPLC analysis: (C18, 10-90% acetonitrile inwater + 0.1% trifluoroacetic acid over 20 min: retention time, % area at254 nm): 12.4 min, 99.4%. 66

1-(3-(9H-carbazol- 9-yl)-2-hydroxy-2- methylpropyl) piperidin-2-one (300MHz, CDCl₃): δ 8.08 (d, 2H, J = 8.1 Hz), 7.54-7.42 (m, 4H), 7.26- 7.20(m, 2H), 4.70 (s, 1H), 4.33 (s, 2H), 4.02 (d, 1H, J = 14.1 Hz), 3.43-3.38 (m, 1H), 3.32-3.26 (m, 1H), 3.19 (d, 1H, J = 13.8 Hz), 2.44 (br t,1H, J = 6.2 Hz), 1.86-1.76 (m, 4H), 1.29 (s, 3H); HPLC analysis: (C18,10-90% acetonitrile in water + 0.1% 337.2 (M + H) trifluoroacetic acidover 20 min: retention time, % area at 254 nm): 12.1 min, 100%. 67

1-(3-(9H-carbazol- 9-yl)-2-hydroxy-2- methylpropyl) pyrrolidin-2-one(300 MHz, CDCl₃): δ 8.08 (d, 2H, J = 7.5 Hz), 7.53-7.42 (m, 4H), 7.26-7.21 (m, 2H), 4.33 (d, 2H, J = 2.4 Hz), 3.59-3.35 (m, 5H), 2.42 (t, 2H,J = 8.3 Hz), 2.11-1.99 (m, 2H), 1.27 (s, 3H); HPLC analysis: (C18, 10-90% acetonitrile in water + 0.1% trifluoroacetic acid over 20 min:retention time, % area at 254 nm): 11.3 min, 100%. 323.0 (M + H) 68

1-(3-(9H-carbazol- 9-yl)-2- hydroxypropyl) pyrrolidin-2-one (300 MHz,CDCl₃): δ 8.09 (d, 2H, J = 7.8 Hz), 7.49-7.42 (m, 4H), 7.29- 7.21 (m,2H), 4.42-4.29 (m, 3H), 3.87 (d, 1H, J = 3.3 Hz), 3.51 (dd, 1H, J = 6.9,14.0 Hz), 3.39-3.20 (m, 3H), 2.40 (t, 2H, J = 8.3 Hz), 1.99 (quin, 2H, J= 7.5 Hz); HPLC analysis: (C18, 10-90% acetonitrile in water + 0.1%trifluoroacetic acid 309.0 (M + H) over 20 min: retention time, % areaat 254 nm): 10.7 min, 100%. 69

1-(3-(9H-carbazol- 9-yl)-2- hydroxypropyl)-3,3- difluoropiperidin-2- one(300 MHz, CDCl₃): δ 8.10-8.07 (m, 2H), 7.47-7.44 (m, 4H), 7.28-7.22 (m,2H), 4.50 (br m, 1H), 4.20-4.38 (d, 2H, J = 6.1 Hz), 3.66-3.51 (m, 2H),3.41-3.30 (m, 2H), 2.77 (d, 1H, J = 3.6 Hz), 2.31-2.18 (m, 2H), 1.96(quin, 2H, J = 6.3 Hz); HPLC analysis: (C18, 10-90% acetonitrile inwater + 0.1% trifluoroacetic acid 359.0 (M + H) over 20 min: retentiontime, % area at 254 nm): 12.1 min, 98.9%. 70

1-(3-(9H-carbazol- 9-yl)-2- hydroxypropyl)-3- fluoropiperidin-2- one(300 MHz, CDCl₃): δ 8.09 (d, 2H, J = 7.8 Hz), 7.50-7.44 (m, 4H), 7.29-7.20 (m, 2H), 4.94-4.71 (m, 1H), 4.45-4.34 (m, 3H), 3.73 (ddd, 1H, J =8.4, 14.4, 34.5 Hz), 3.57-3.07 (m, 4H), 2.15-1.88 (m, 3H), 1.77-1.66 (m,1H); HPLC analysis: (C18, 10- 90% acetonitrile in water + 0.1%trifluoroacetic acid over 20 min: 341.2 (M + H) retention time, % areaat 254 nm): 11.3 min, 100%.

Compound 71:1-(3-(3,6-difluoro-9H-carbazol-9-yl)-2-hydroxypropyl)tetrahydropyrimidin-2(1H)-one

A mixture of 3,6-difluoro-9-(oxiran-2-ylmethyl)-9H-carbazole (0.06 g,0.2 mmol) and 1,3-diaminopropane (0.195 mL, 2.3 mmol) in ethanol (2 mL)was stirred at 40° C. for 5 hrs. The reaction mixture was concentratedin vacuo to afford a light yellow oil. ESI (m/z): 334.2 (M+H). Theresidue was dissolved in methylene chloride (10 mL) at 0° C. were added4-dimethylaminopyridine (0.005 g) and 1,1′-carbonyldiimidazole (0.056 g,0.3 mmol). The mixture was warmed to room temperature and stirred for 16hrs. The reaction was concentrated in vacuo and the residue purified bysilica gel chromatography (0-10% methanol/methylene chloride) to affordthe desired product as a white powder (0.060 g, 72%). ¹H NMR (300 MHz,d⁶-DMSO): δ 7.99 (dd, 2H, J=9.6, 2.7 Hz), 7.56 (dd, 2H, J=9.0, 4.2 Hz),7.30 (td, 2H, J=9.0, 2.7 Hz), 6.29 (s, 1H), 5.24 (d, 1H, J=5.4 Hz), 4.35(dd, 1H, J=15.0, 3.6 Hz), 4.24 (dd, 1H, J=15.0, 8.1 Hz), 4.07 (m, 1H),3.55-3.05 (m, 6H), 1.90-1.70 (m, 2H); ESI (m/z): 360.1 (M+H).

Compounds 72 to 84

Compounds 72 to 84 were prepared by procedures analogous to those usedfor Compound 71 using an appropriate diamine such asN-methyl-1,3-diaminopropane, ethylenediamine, N-ethylethlenediamine,N1-cyclohexylpropane-1,3-diamine, N1-cyclobutylpropane-1,3-diamine,N1-cyclopentylpropane-1,3-diamine or propane-1,2-diamine instead of1,3-diaminopropane. Preparation of non-commercially available diaminesused are described in the preparation of intermediates.

Cpd ESI # Structure Name ¹H NMR (m/z) 72

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxypropyl)imidazolidin-2-one (300 MHz, CDCl₃): δ 7.69-7.65 (dd, 2H, J = 2.4, 8.7Hz), 7.40-7.36 (dd, 2H, J = 4.0, 9.0 Hz), 7.24-7.18 (td, 2H, J = 2.7,9.0 Hz), 4.42-4.35 (m, 4H), 4.24-4.22 (m, 1H), 3.50-3.21 (m, 6H). 346.1(M + H) 73

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxypropyl)-3-ethylimidazolidin-2- one (300 MHz, CDCl₃): δ 7.68-7.64 (dd, 2H, J = 2.4,8.7 Hz), 7.39-7.35 (dd, 2H, J = 4.2, 9.0 Hz), 7.23-7.17 (td, 2H, J =2.7, 8.7 Hz), 4.80 (br s, 1H), 4.40-4.30 (m, 3H), 3.34-3.07 (m, 8H),1.15-1.10 (t, 3H, J = 7.2 Hz). 373.4 (M + H) 74

1-cyclohexyl-3-(3- (3,6-difluoro-9H- carbazol-9-yl)-2- hydroxypropyl)tetrahydropyrimidin- 2(1H)-one (300 MHz, CDCl₃): δ 7.67-7.64 (dd, 2H, J= 2.0, 8.4 Hz), 7.40-7.36 (dd, 2H, J = 4.1, 9.0 Hz), 7.23-7.16 (td, 2H,J = 2.4, 9.0 Hz), 5.66-5.65 (d, 1H, J = 2.1 Hz), 4.40-4.17 (m, 4H),3.88-3.81 (m, 1H), 3.21-2.94 (m, 4H), 2.85-2.80 (dd, 1H, J = 1.7, 14.4Hz), 1.85-1.61 (m, 7H), 1.45-1.26 (m, 4H), 1.10-1.01 (m, 1H). 442.1 (M +H) 75

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxypropyl)-3-phenyltetrahydro- pyrimidin-2(1H)- one (300 MHz, CDCl₃): δ 7.68-7.64(dd, 2H, J = 2.7, 8.7 Hz), 7.41-7.36 (dd, 2H, J = 4.1, 8.7 Hz),7.34-7.32 (m, 2H), 7.24-7.16 (m, 5H), 5.07-5.06 (d, 1H, J = 3.0 Hz),4.40-4.26 (m, 3H), 3.93-3.85 (dd, 1H, J = 8.7, 14.7 Hz), 3.67-3.63 (m,2H), 3.26-3.18 (m, 2H), 3.00-3.95 (dd, 1H, J = 2.0, 14.6 Hz), 2.10-1.99(m, 2H). 436.2 (M + H) 76

1-cyclopentyl-3-(3- (3,6-difluoro-9H- carbazol-9-yl)-2- hydroxypropyl)tetrahydropyrimidin- 2(1H)-one (300 MHz, CDCl₃): δ 7.67-7.63 (dd, 2H, J= 2.4, 8.7 Hz), 7.40-7.35 (dd, 2H, J = 4.2, 8.7 Hz), 7.23-7.16 (td, 2H,J = 2.4, 9.0 Hz), 5.65-5.65 (d, 1H, J = 2.4 Hz), 4.84-4.73 (quint, 1H, J= 8.4 Hz), 4.40-4.21 (m, 3H), 3.88-3.81 (m, 1H), 3.18-2.95 (m, 4H),2.85-2.80 (dd, 1H, J = 1.5, 14.7 Hz), 1.88-1.34 (m, 10H). 428.2 (M + H)77

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxypropyl)-3-isopropyltetrahydro- pyrimidin-2(1H)-one (300 MHz, CDCl₃): δ 7.67-7.64(dd, 2H, J = 2.1, 8.9 Hz), 7.40-7.35 (dd, 2H, J = 4.2, 9.0 Hz),7.23-7.17 (td, 2H, J = 2.6, 9.0 Hz), 5.65-5.64 (d, 1H, J = 2.4 Hz),4.74-4.60 (sept, 1H, J = 6.9 Hz), 4.41-4.21 (m, 3H), 3.91- 3.82 (m, 1H),3.18-2.84 (m, 4H), 2.85-2.79 (dd, 1H, J = 1.7, 14.6 Hz), 1.90-1.75 (m,2H), 1.09-1.07 (d, 6H, J = 6.9 Hz); HPLC analysis: (C18, 10-90%acetonitrile in water over 20 min: retention time, % area at 254 nm):13.5 min, 100%. 402.1 (M + H) 78

1-cyclobutyl-3-(3- (3,6-difluoro-9H- carbazol-9-yl)-2- hydroxypropyl)tetrahydropyrimidin- 2(1H)-one (300 MHz, CDCl₃): δ 7.67-7.64 (dd, 2H, J= 2.4, 8.7 Hz), 7.39-7.35 (dd, 2H, J = 4.2, 8.7 Hz), 7.24-7.16 (td, 2H,J = 2.2, 8.7 Hz), 5.46-5.45 (d, 1H, J = 1.8 Hz), 4.90-4.79 (quint, 1H, J= 8.7 Hz), 4.41-4.21 (m, 3H), 3.86-3.79 (m, 1H), 3.31-2.99 (m, 5H),2.87-2.81 (dd, 1H, J = 1.5, 14.4 Hz), 2.09-2.02 (m, 4H), 1.90-1.83 (m,2H), 1.67-1.60 (m, 2H). 414.2 (M + H) 79

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)- 2-hydroxy-2- methylpropyl)-3-phenyltetrahydro- pyrimidin-2(1H)- one (300 MHz, CDCl₃): δ 7.64-7.60(dd, 2H, J = 2.7, 8.7 Hz), 7.46-7.42 (dd, 2H, J = 4.1, 9.0 Hz),7.35-7.30 (m, 2H), 7.23-7.13 (m, 5H), 5.48 (s, 1H), 4.27-4.26 (d, 2H, J= 3.0 Hz,) 4.00- 3.95-3.86, 3.13-3.08 (ABq, 2H, J = 14.6 Hz), 3.72-3.46(m, 4H), 2.18- 2.05 (m, 2H), 1.33 (s, 3H). 450.1 (M + H) 80

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)- 2-hydroxy-2- methylpropyl)-3-isopropyltetrahydro- pyrimidin-2(1H)- one (300 MHz, CDCl₃): δ 7.65-7.61(dd, 2H, J = 2.7, 8.7 Hz), 7.47-7.43 (dd, 2H, J = 4.2, 9.0 Hz),7.20-7.13 (td, 2H, J = 2.6, 9.0 Hz), 6.07 (s, 1H), 4.70-4.60 (sept, 1H,J = 6.9 Hz), 4.25-4.21 (d, 2H, J = 5.1 Hz), 3.92- 3.87, 3.03-2.98 (ABq,2H, J = 14.4 Hz), 3.39-3.10 (m, 4H), 1.98-1.90 (m, 2H), 1.27 (s, 3H).416.1 (M + H) 81

1-cyclobutyl-3-(3- (3,6-difluoro-9H- carbazol-9-yl)-2- hydroxy-2-methylpropyl) tetrahydropyrimidin- 2(1H)-one (300 MHz, CDCl₃): δ7.65-7.61 (dd, 2H, J = 2.6, 8.6 Hz), 7.47-7.42 (dd, 2H, J = 4.1, 8.9Hz), 7.20-7.13 (td, 2H, J = 2.5, 9.0 Hz), 5.91 (s, 1H), 4.87-4.76(quint, 1H, J = 8.6 Hz), 4.25-4.24 (d, 2H, J = 4.5 Hz), 3.92- 3.87,3.12-2.96 (ABq, 2H, J = 14.7 Hz), 3.42-3.21 (m, 4H), 2.13-1.91 (m, 6H),1.66-1.55 (m, 2H) 1.26 (s, 3H). 428.1 (M + H) 82

1-cyclohexyl-3-(3- (3,6-difluoro-9H- carbazol-9-yl)-2- hydroxy-2-methylpropyl) tetrahydropyrimidin- 2(1H)-one (300 MHz, CDCl₃): δ7.64-7.60 (dd, 2H, J = 2.7, 8.7 Hz), 7.47-7.43 (dd, 2H, J = 4.2, 9.0Hz), 7.20-7.13 (td, 2H, J = 2.6, 9.0 Hz), 6.07 (s, 1H), 4.25-4.24 (d,2H, J = 3.6 Hz), 4.20- 4.16 (m, 1H), 3.91-3.87, 3.02-2.97 (ABq, 2H, J =14.6 Hz), 3.41-3.10 (m, 4H), 1.97-1.62 (m, 6H), 1.38- 1.27 (m, 3H) 1.26(s, 3H), 1.07-1.00 (m, 1H). 456.2 (M + H) 83

1-cyclopropyl-3-(3- (3,6-difluoro-9H- carbazol-9-yl)-2- hydroxypropyl)tetrahydropyrimidin- 2(1H)-one (300 MHz, CDCl₃): δ 7.66-7.63 (dd, 2H, J= 2.3, 9.0 Hz), 7.39-7.34 (dd, 2H, J = 4.2, 9.0 Hz), 7.22-7.19 (td, 2H,J = 2.6, 9.0 Hz) 5.24 (s 1H), 4.39-4.21 (m, 3H), 3.84-3.76 (m, 1H),3.25-3.19 (m, 2H), 3.11-2.95 (m, 2H), 2.92-2.86 (m, 1H), 2.61- 2.54 (m,1H), 1.87-1.79 (quint, 2H, J = 6.0 Hz), 0.78-0.65 (m, 2H), 0.64- 0.57(m, 2H). 400.2 (M + H) 84

1-(3-(9H-carbazol- 9-yl)-2- hydroxypropyl) imidazolidin-2-one (300 MHz,d⁶-DMSO): δ 8.11 (d, 2H, J = 7.5 Hz), 7.57 (d, 2H, J = 8.1 Hz),7.44-7.38 (m, 2H), 7.16 (t, 2H, J = 7.1 Hz), 6.30 (s, 1H), 5.16 (d, 1H,J = 6.0 Hz), 4.34 (dd, 1H, J = 4.8, 14.7 Hz), 4.25 (dd, 1H, J = 7.5,15.0 Hz), 4.09-4.02 (m, 1H), 3.45- 3.17 (m, 5H), 3.10 (dd, 1H, J = 7.2,13.8 Hz); HPLC analysis: (C18, 10- 90% acetonitrile in water + 0.1%310.0 (M + H) trifluoroacetic acid over 20 min: retention time, % areaat 254 nm): 10.1 min, 100%.

Compound 85:1-(3-(3,6-difluoro-9H-carbazol-9-yl)-2-hydroxypropyl)-4-methylimidazolidin-2-one

A mixture of tert-butyl(1-((3-(3,6-difluoro-9H-carbazol-9-yl)-2-hydroxypropyl)amino)propan-2-yl)carbamate(1.400 g, 80% pure, 2.6 mmol, 1.0 equiv.) and potassium tert-butoxide(0.290 g, 2.6 mmol, 1.0 equiv.) in anhydrous tetrahydrofuran (270 mL)was stirred at reflux for 2 hrs. The reaction mixture was cooled to roomtemperature, acetic acid (0.1 mL) and silica gel were added, and themixture concentrated under reduced pressure to a slurry. The cruderesidue was purified by silica gel column chromatography by eluting witha gradient of 0-10% methanol/methylene chloride to give a solid. Thesolid was crystallized from ethyl acetate to give a white solid (0.673g, 72%). ¹H NMR (300 MHz, CDCl₃; mixture of diastereomers): δ 7.69-7.65(dd, 2H, J=2.7, 8.7 Hz), 7.40-7.35 (dd, 2H, J=4.1, 9.0 Hz), 7.24-7.18(td, 2H, J=2.7, 9.0 Hz), 4.47-4.19 (m, 5H), 3.82 (br m, 1H), 3.58-2.93(m, 4H), 1.26-1.24 (d, 3H, J=6.3 Hz); ESI (m/z): 360.9 (M+H).

Compound 86:1-(3-(3,6-difluoro-9H-carbazol-9-yl)-2-hydroxypropyl)-3-ethyltetrahydropyrimidin-2(1H)-one

To a solution of 3,6-difluoro-9H-carbazole (0.065 g, 0.3 mmol) inanhydrous N,N-dimethylformamide (0.3 mL) was added 60% sodium hydride inmineral oil (0.009 g, 0.2 mmol) and the mixture stirred at roomtemperature for 30 min. A solution of1-ethyl-3-(oxiran-2-ylmethyl)tetrahydropyrimidin-2(1H)-one (0.055 g, 0.3mmol) in anhydrous N,N-dimethylformamide (0.3 mL) was added and themixture was heated at 70° C. for 8 hrs. The reaction mixture was dilutedwith methanol, filtered, and purified by preparative HPLC (C18, 30-95%acetonitrile in water) to afford a white solid (0.072 g, 62%). ¹H NMR(300 MHz, CDCl₃): δ 7.67-7.63 (dd, 2H, J=2.6, 9.0 Hz), 7.39-7.35 (dd,2H, J=4.2, 8.7 Hz), 7.23-7.16 (td, 2H, J=2.4, 9.0 Hz), 5.52 (d, 1H,J=2.7 Hz), 4.37-4.21 (m, 3H), 3.86-3.79 (m, 1H), 3.43-2.98 (m, 6H),2.88-2.83 (dd, 1H, J=1.8, 14.7 Hz), 1.91-1.83 (quin, 2H, J=5.9 Hz),1.12-1.07 (t, 3H, J=7.4 Hz); ESI (m/z): 388.1 (M+H); HPLC analysis:(C18, 10-90% acetonitrile in water over 20 min: retention time, % areaat 254 nm): 12.8 min, 100%.

Compounds 87-100

Compounds 87-100 were prepared by procedures analogous to those used forCompound 88.

Cpd ESI # Structure Name ¹H NMR (m/z) 87

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)- 2-hydroxy-2- methylpropyl)-3-ethyltetrahydro- pyrimidin-2(1H)- one (300 MHz, CDCl₃): δ 7.64-7.61 (dd,2H, J = 2.5, 8.6 Hz), 7.47-7.42 (dd, 2H, J = 4.2, 9.0 Hz), 7.20-7.13(td, 2H, J = 2.7, 9.0 Hz), 5.97 (s, 1H), 4.30-4.18 (m, 2H,) 3.91-3.86,3.03- 2.98 (ABq, 2H, J = 14.9 Hz), 3.46- 3.23 (m, 6H), 1.99-1.93 (m,2H), 1.26 (s, 3H), 1.12-1.07 (t, 3H, J = 7.0 Hz). 402.1 (M + H) 88

1-cyclohexyl-3-(3- (3,6-difluoro-9H- carbazol-9-yl)-2 hydroxypropyl)imidazolidin-2-one (300 MHz, CDCl₃): δ 7.68-7.64 (dd, 2H, J = 2.6, 8.6Hz), 7.39-7.35 (dd, 2H, J = 4.2, 8.7 Hz), 7.39-7.17 (td, 2H, J = 2.4,9.0 Hz), 5.14-5.13 (d, 1H, J = 2.7 Hz), 4.40-4.30 (m, 3H,) 3.73-3.64 (m,1H), 3.30-2.96 (m, 6H), 1.80-1.70 (m, 5H), 1.45-1.24 (m, 4H), 1.13-1.05(m, 1H). 428.2 (M + H) 89

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)- 2-hydroxypropyl)-3-phenylimidazolidin- 2-one (300 MHz, CDCl₃): δ 7.69-7.66 (dd, 2H, J =2.6, 8.6 Hz), 7.53-7.50 (m, 2H), 7.41-7.32 (m, 4H), 7.39-7.17 (td, 2H, J= 2.5, 9.0 Hz), 7.10-7.05 (br t, 1H, J = 7.4 Hz), 4.43-4.37 (m, 3H),4.01-3.99 (d, 1H, J = 4.2 Hz) 3.86-3.80 (m, 2H), 3.53-3.29 (m, 4H).422.1 (M + H) 90

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)- 2-hydroxypropyl)-3- isopropyl-imidazolidin-2-one (300 MHz, CDCl₃): δ 7.66-7.62 (dd, 2H, J = 2.4, 8.6Hz), 7.38-7.33 (dd, 2H, J = 4.1, 8.9 Hz), 7.22-7.15 (td, 2H, J = 2.5,9.0 Hz), 5.00-5.00 (d, 1H, J = 2.1 Hz), 4.37-4.06 (m, 3H), 4.15-4.06 (m,1H), 3.29-3.00 (m, 6H), 1.15-1.11 (t, 6H, J = 6.3 Hz). 388.2 (M + H) 91

1-cyclopentyl-3-(3- (3,6-difluoro-9H- carbazol-9-yl)-2- hydroxypropyl)imidazolidin-2-one (300 MHz, CDCl₃): δ 7.66-7.65 (dd, 2H, J = 2.6, 8.6Hz), 7.40-7.35 (dd, 2H, J = 4.2, 8.7 Hz), 7.24-7.17 (td, 2H, J = 2.6,9.0 Hz), 4.98-4.97 (d, 1H, J = 1.5 Hz), 4.41-4.22 (m, 3H), 3.32-3.00 (m,7H), 1.86-1.48 (m, 10H). 414.2 (M + H) 92

1-cyclopropyl-3-(3- (3,6-difluoro-9H- carbazol-9-yl)-2- hydroxypropyl)imidazolidin-2-one (300 MHz, CDCl₃): δ 7.66-7.62 (dd, 2H, J = 2.6, 8.9Hz), 7.38-7.34 (dd, 2H, J = 4.1, 8.9 Hz), 7.22-7.15 (td, 2H, J = 2.5,9.0 Hz), 4.81-4.80 (d, 1H, J = 4.5 Hz), 4.37-4.28 (m, 3H), 3.31-3.00 (m,6H), 2.44-2.37 (m, 1H), 0.76-0.61 (m, 4H). 386.2 (M + H) 93

1-cyclobutyl-3-(3- (3,6-difluoro-9H- carbazol-9-yl)-2- hydroxypropyl)imidazolidin-2-one (300 MHz, CDCl₃): δ 7.66-7.62 (dd, 2H, J = 2.6, 9.0Hz), 7.37-7.33 (dd, 2H, J = 4.1, 9.0 Hz), 7.22-7.15 (td, 2H, J = 2.5,9.0 Hz), 4.80-4.79 (d, 1H, J = 4.5 Hz), 4.43-4.27 (m, 4H), 3.40-3.04 (m,6H), 2.16-2.04 (m, 4H), 1.71-1.60 (m, 2H). 400.1 (M + H) 94

1-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxypropyl)-3,5-dimethyltetrahydro- pyrimidin-2(1H)-one (300 MHz, CDCl₃): δ 7.68-7.64(dd, 2H, J = 2.7, 8.7 Hz), 7.40-7.36 (dd, 2H, J = 4.2, 9.0 Hz),7.23-7.17 (td, 2H, J = 2.6, 9.0 Hz), 5.31 (br s, 0.5H), 5.15 (br s,0.3H), 4.39-4.21 (m, 3H), 3.85-3.73 (m, 1H), 3.17- 2.76 (m, 8H),2.17-2.12 (m, 1H), 0.93-0.90 (two overlapping d, 3H, J = 6.9 Hz). 388.2(M + H) 95

1-(3-(9H-carbazol- 9-yl)-2- hydroxypropyl)-3- ethyltetrahydro-pyrimidin-2(1H)-one (300 MHz, CDCl₃): δ 8.08 (d, 2H, J = 8.1 Hz),7.49-7.42 (m, 4H), 7.26- 7.21 (m, 2H), 5.44 (d, 1H, J = 3.0 Hz),4.43-4.25 (m, 3H), 3.87 (dd, 1H, J = 14.1, 8.6 Hz), 3.45-2.84 (m, 7H),1.82 (quin, 2H, J = 6.0 Hz), 1.09 (t, 3H, J = 7.1 Hz); HPLC analysis:(C18, 10-95% acetonitrile in water + 0.1% trifluoroacetic acid over 15min: retention time, % area 352.2 (M + H) at 254 nm): 12.2 min, 98.3%.96

1-(3-(9H-carbazol- 9-yl)-2- hydroxypropyl)-3- cyclobutyl-imidazolidin-2-one (300 MHz, CDCl₃): δ 8.11-8.07 (m, 2H), 7.48-7.42 (m,4H), 7.28-7.21 (m, 2H), 4.74-4.73 (m, 1H), 4.45- 4.34 (m, 4H), 3.35 (t,2H, J = 7.8 Hz), 3.24-2.99 (m, 4H), 2.17-2.07 (m, 4H), 1.71-1.61 (m,2H); HPLC analysis: (C18, 10-95% acetonitrile in water + 0.1%trifluoroacetic acid over 15 min: retention time, % area at 254 nm):12.7 min, 95.1%. 365.0 (M + H) 97

1-(3-(9H-carbazol- 9-yl)-2- hydroxypropyl)-3- cyclopropyl-imidazolidin-2-one (300 MHz, CDCl₃): δ 8.09 (d, 2H, J = 7.2 Hz),7.48-7.42 (m, 4H), 7.27- 7.21 (m, 2H), 4.75 (br s, 1H), 4.45- 4.35 (m,3H), 3.28-2.94 (m, 6H), 2.45-2.40 (m, 1H), 0.75-0.62 (m, 4H); HPLCanalysis: (C18, 10-95% acetonitrile in water + 0.1% trifluoroacetic acidover 15 min: retention time, % area at 254 nm): 11.6 min, 96.2%. 351.0(M + H) 98

1-(3-(9H-carbazol- 9-yl)-2- hydroxypropyl)-3- isopropyl-imidazolidin-2-one (300 MHz, CDCl₃): δ 8.09 (d, 2H, J = 7.8 Hz),7.49-7.42 (m, 4H), 7.27- 7.21 (m, 2H), 4.96-4.94 (m, 1H), 4.48-4.35 (m,3H), 4.13 (sept, 1H, J = 6.6 Hz), 3.26-2.94 (m, 6 H), 1.15 (d, 3H, J =7.5 Hz), 1.12 (d, 3H, J = 7.2 Hz); HPLC analysis: (C18, 10- 95%acetonitrile in water + 0.1% trifluoroacetic acid over 15 min: retentiontime, % area at 254 nm): 353.0 (M + H) 11.6 min, 98.3%. 99

1-(3-(9H-carbazol- 9-yl)-2- hydroxypropyl)-3- cyclopropyltetra-hydropyrimidin- 2(1H)-one (300 MHz, CDCl₃): δ 8.09 (d, 2H, J = 7.5 Hz),7.48-7.42 (m, 4H), 7.26- 7.21 (m, 2H), 5.25 (d, 1H, J = 3.0 Hz),4.45-4.26 (m, 3H), 3.91-3.83 (dd, 1H, J = 9.0, 14.7 Hz), 3.21-3.17 (m,2H), 3.04-2.85 (m, 3H), 2.62- 2.56 (m, 1H), 1.78 (quint, 2H, J = 6.0Hz), 0.88-0.57 (m, 4H); HPLC analysis: (C18, 10-95% acetonitrile inwater + 0.1% trifluoroacetic acid over 15 min: retention time, % area at254 nm): 12.1 min, 97.5%. 364.1 (M + H) 100 

1-(3-(9H-carbazol- 9-yl)-2- hydroxypropyl)-3- cyclobutyltetra-hydropyrimidin- 2(1H)-one (300 MHz, CDCl₃): δ 8.10-8.07 (m, 2H),7.48-7.42 (m, 4H), 7.26-7.21 (m, 2H), 5.41 (d, 1H, J = 2.4 Hz), 4.85(quint, 1H, J = 9.0 Hz), 4.45- 4.25 (m, 3H), 3.87 (dd, 1H, J = 8.9, 14.6Hz), 3.35-3.11 (m, 2H), 3.03- 2.87 (m, 3H), 2.10-2.00 (m, 4H), 1.85-1.77(m, 2H), 1.66-1.58 (m, 2H); HPLC analysis: (C18, 10-95% acetonitrile inwater + 0.1% 378.2 (M + H) trifluoroacetic acid over 15 min: retentiontime, % area at 254 nm): 13.3 min, 95.6%.

Compound 101:1-(3-(3,6-difluoro-9H-carbazol-9-yl)-2-hydroxypropyl)-3,4-dimethyltetrahydropyrimidin-2(1H)-one

To a stirred solution of 3,6-difluoro-9H-carbazole (0.38 g, 1.9 mmol,1.2 equiv.) in anhydrous N,N-dimethylacetamide (2 mL) was added 60%sodium hydride in mineral oil (0.075 g, 1.9 mmol, 1.2 equiv.). Afterstirring at room temperature for 2 hrs, the resulting carbazole sodiumsolution was ready for use.

To a stirred solution of 1,6-dimethyltetrahydropyrimidin-2(1H)-one (0.2g, 1.6 mmol, 1.0 equiv.) in dry N,N-dimethylacetamide (3 mL) at 0° C.was added 60% sodium hydride in mineral oil (0.075 g, 1.9 mmol, 1.2equiv.) and the mixture was stirred for 1 hr at room temperature.Epibromohydrin (0.155 mL, 1.9 mmol, 1.2 equiv.) was added at 0° C. andthe mixture was slowly warmed to room temperature and stirred for 16hrs. The carbazole sodium solution was added and the mixture was heatedat 70° C. for 5 hrs. The reaction was diluted with water and extractedwith ethyl acetate. The organic layers were washed with saturatedaqueous sodium chloride, dried over sodium sulfate, filtered andconcentrated in vacuo. The residue was purified by silica gel column(0-100% ethyl acetate/hexane) to afford the pure product as a white foam(0.116 g, 19%). ¹H NMR (300 MHz, CDCl₃): (a mixture of twodiastereomers) δ 7.68 (dd, 2H, J=8.7, 2.7 Hz), 7.40 (dd, 2H, J=9.0, 4.2Hz), 7.27-7.18 (m, 2H), 5.51 and 5.06 (d, 1H, J=2.7 Hz), 4.50-4.20 (m,3H), 3.90-3.74 (m, 1H), 3.50-2.75 (m, 7H), 2.05 (m, 1H), 1.60 (m, 1H),1.21 and 1.17 (d, 3H, J=6.6 Hz); ESI (m/z): 388.2 (M+H).

Compound 102:3-(3-(3,6-difluoro-9H-carbazol-9-yl)-2-hydroxy-2-methylpropyl)-3,4-dihydroquinazolin-2(1H)-one

A mixture of1-benzyl-3-(3-(3,6-difluoro-9H-carbazol-9-yl)-2-hydroxy-2-methylpropyl)-3,4-dihydroquinazolin-2(1H)-one(0.102 g, 0.2 mmol, 1.0 equiv.) and 20% palladium hydroxide on carbon(0.035 g) in acetic acid (4 mL) and tetrahydrofuran (2 mL) was stirredunder 50 psi of hydrogen for 48 hrs. 20% palladium hydroxide on carbon(0.02 g) and 10% palladium on carbon (0.01 g) were added and thereaction mixture stirred under 50 psi of hydrogen for 72 hrs. Themixture was filtered, concentrated, and purified by preparative HPLC(C18, 40-80% acetonitrile in water) to give a white solid (0.013 g,15%). ¹H NMR (300 MHz, CD₃OD): δ 7.73-7.70 (dd, 2H, J=2.6, 8.6 Hz),7.55-7.51 (dd, 2H, J=4.1, 9.2 Hz), 7.27-7.05 (m, 3H), 7.07-7.05 (d, 1H,J=7.2 Hz), 6.96-6.91 (td, 1H, J=1.1, 7.5 Hz), 6.81-6.78 (d, 1H, J=8.1Hz), 4.71 (s, 2H), 4.33 (s, 2H) 3.75-3.70, 3.59-3.54 (ABq, 2H, J=14.4Hz), 1.22 (s, 3H); ESI (m/z): 422.1 (M+H); HPLC analysis: (C18, 5-95%acetonitrile in water+0.1% trifluoroacetic acid over 20 min: retentiontime, % area at 254 nm): 13.5 min, 91.5%.

The following compound was prepared by procedures analogous to thoseused for Compound 102.

Cpd ESI # Structure Name ¹H NMR & HPLC (m/z) 103

3-(3-(3,6-difluoro- 9H-carbazol-9-yl)-2- hydroxypropyl)-3,4-dihydroquinazolin- 2(1H)-one (300 MHz, CD₃OD): δ 7.75-7.71 (dd, 2H, J =2.4, 8.7 Hz), 7.51-7.47 (dd, 2H, J = 4.2, 8.9 Hz), 7.20-7.11 (m, 3H),7.04-7.02 (m, 1H), 6.94- 6.89 (td, 1H, J = 1.1, 7.2 Hz), 6.78- 6.76 (d,1H, J = 7.8 Hz), 4.67-4.62, 4.58-4.53 (ABq, 2H, J = 14.1 Hz), 4.42-4.32(m, 3H), 3.81-3.75 (dd, lH, J = 3.9, 14.1 Hz), 3.41-3.34 (dd, 1H, J =4.2, 14.1 Hz); HPLC analysis: (C18, 5-95% acetonitrile in water + 0.1%trifluoroacetic acid 408.2 (M + H) over 20 min: retention time, % areaat 254 nm): 12.8 min, 96 %.

Compound 104:(1S,4R)-2-((R)-3-(3,6-difluoro-9H-carbazol-9-yl)-2-hydroxypropyl)-2-azabicyclo[2.2.1]heptan-3-one

To a stirred solution of (1S,4R)-2-azabicyclo[2.2.1]heptan-3-one (0.051g, 0.5 mmol, 2.0 equiv.) in anhydrous tetrahydrofuran (1.5 mL) was added60% sodium hydride in mineral oil (0.009 g, 0.2 mmol, 1.0 equiv.) andthe mixture was stirred at room temperature for 60 mins.(R)-3,6-Difluoro-9-(oxiran-2-ylmethyl)-9H-carbazole (0.060 g, 0.2 mmol,1.0 equiv.) was added and the mixture was stirred at 80° C. in a sealedtube for 16 hrs. The mixture was cooled to room temperature and 1 Nacetic acid (0.02 mL) in methanol was added and then concentrated underreduced pressure. The crude residue was purified by preparative HPLC(C18, 30-95% acetonitrile in water) to give a white solid (0.053 g,61%). ¹H NMR (300 MHz, CDCl₃): δ 7.68-7.65 (dd, 2H, J=2.6, 8.6 Hz),7.38-7.33 (dd, 2H, J=4.1, 8.9 Hz), 7.24-7.17 (td, 2H, J=2.5, 9.0 Hz),4.38-4.28 (m, 4H), 3.64 (br s, 1H), 3.43-3.38 (dd, 1H, J=2.7, 14.1 Hz),3.02-2.96 (dd, 1H, J=2.7, 14.4 Hz), 2.90-2.89 (m, 1H), 1.96-1.40 (m,6H); ESI (m/z): 371.1 (M+H); HPLC analysis: (C18, 5-95% acetonitrile inwater+0.1% trifluoroacetic acid over 20 min: retention time, % area at254 nm): 12.2 min, 100%; Chiral HPLC analysis (Chiralcel AD-H, 15%ethanol in hexanes over 42 mins: retention time, % area at 254 nm): 7.7min, 98.5%; 12.6 min, 0.7% (98.5% de).

Compounds 105-138

Compounds 105-138 were prepared by procedures analogous to those usedfor Compound 104.

Cpd ESI # Structure Name ¹H NMR & HPLC (m/z) 105

(1R,4S)-2-((R)-3- (3,6-difluoro-9H- carbazol-9-yl)-2- hydroxypropyl)-2-azabicyclo[2.2.1] heptan-3-one (300 MHz, CDCl₃): δ 7.69-7.65 (dd, 2H, J= 2.6, 8.7 Hz), 7.38-7.33 (dd, 2H, J = 3.9, 9.0 Hz), 7.25-7.19 (td, 2H,J = 2.6, 9.0 Hz), 4.40-4.23 (m, 4H), 3.73 (br s, 1H), 3.48-3.41 (dd, 1H,J = 7.4, 14.1 Hz), 2.95-2.90 (dd, 1H, J = 1.7, 13.7 Hz), 2.86-2.85 (dd,1H, J = 1.2, 3.9 Hz), 1.92-1.36 (m, 6 H); HPLC analysis: (C18, 5-95%acetonitrile in water + 0.1% trifluoroacetic acid over 20 min: retentiontime, % area at 254 nm): 371.1 (M + H) 12.2 min, 100%; Chiral HPLCanalysis (Chiralcel AD-H, 15% ethanol in hexanes over 42 mins: retentiontime, % area at 254 nm): 21.1 min, 97.6%; 39.0 min, 2.3% (95.2% de). 106

(1S,4R)-2-((R)-3- (9H-carbazol-9-yl)- 2-hydroxypropyl)-2-azabicyclo[2.2.1] heptan-3-one (300 MHz, CDCl₃): δ 8.10-8.07 (m, 2H),7.46-7.44 (m, 4H), 7.26-7.21 (m, 2H), 4.35-4.34 (m, 4H), 3.61 (br s,1H), 3.41-3.36 (m, 1H), 3.02-2.95 (m, 1H), 2.88-2.86 (m, 1H), 1.97- 1.55(m, 4H), 1.39-1.36 (br d, 1H, J = 10.2 Hz); HPLC analysis: (C18, 5-95%acetonitrile in water + 0.1% trifluoroacetic acid over 20 min: retentiontime, % area at 254 nm): 335.2 (M + H) 11.5 min, 100%; Chiral HPLCanalysis (Chiralcel AD-H, 15% ethanol in hexanes over 30 mins: retentiontime, % area at 254 nm): 9.2 min, 98.1%; 12.0 min, 1.7% (96.4% de). 107

(1R,4S)-2-((R)-3- (9H-carbazol-9-yl)- 2-hydroxypropyl)-2-azabicyclo[2.2.1] heptan-3-one (300 MHz, CDCl₃): δ 8.12-8.09 (dd, 2H,J = 0.9, 7.8 Hz), 7.51-7.43 (m, 4H), 7.28-7.23 (m, 2H), 4.47-4.26 (m,4H), 3.69 (s, 1H), 3.50-3.43 (s, dd, 1H, J = 7.5, 13.8 Hz), 2.95-2.91(d, 1H, J = 14.1 Hz), 2.84-2.83 (d, 1H, J = 3.6 Hz), 1.89-1.31 (m, 6H);HPLC analysis: (C18, 5-95% acetonitrile in water + 0.1% trifluoroaceticacid over 20 min: 335.2 (M + H) retention time, % area at 254 nm): 11.6min, 98.5%; Chiral HPLC analysis (Chiralcel AD-H, 15% ethanol in hexanesover 30 mins: retention time, % area at 254 nm): 9.7 min, 99.0%; 15.3min, 0.3% (99.4% de). 108

(1R,4S)-2-((S)-3- (9H-carbazol-9-yl)- 2-hydroxypropyl)-2-azabicyclo[2.2.1] heptan-3-one (300 MHz, CDCl₃): δ 8.11-8.07 (m, 2H),7.47-7.45 (m, 4H), 7.26-7.21 (m, 2H), 4.35-4.34 (m, 4H), 3.62 (br s,1H), 3.42-3.37(m, 1H), 3.02-2.95 (m, 1H), 2.88-2.86 (m, 1H), 1.98- 1.55(m, 4H), 1.40-1.36 (br d, 1H, J = 9.6 Hz); HPLC analysis: (C18, 5-95%acetonitrile in water + 0.1% trifluoroacetic acid over 20 min: retentiontime, % area at 254 nm): 335.2 (M + H) 11.5 min, 100%; Chiral HPLCanalysis (Chiralcel AD-H, 15% ethanol in hexanes over 30 mins: retentiontime, % area at 254 nm): 9.9 min, 1.5%, 15.2 min, 98.2% (96.9% de). 109

(R)-1-((R)-3-(3,6- difluoro-9H- carbazol-9-yl)-2- hydroxypropyl)-5-methylpyrrolidin-2- one (300 MHz, CDCl₃): δ 7.69-7.65 (dd, 2H, J = 2.6,8.6 Hz), 7.38-7.34 (dd, 2H, J = 3.9, 9.3 Hz), 7.25-7.18 (td, 2H, J =2.6, 9.3 Hz), 4.38-4.28 (m, 3H), 4.07-4.06 (d, 1H, J = 3.0 Hz),3.69-3.56 (m, 2H), 3.08-3.04 (d, 1H, J = 13.5 Hz), 2.46-2.18 (m, 3H),1.66-1.55 (m, 1H), 1.04-1.02 (d, 3H, J = 6.0 Hz); HPLC analysis: (C18,5- 95% acetonitrile in water + 0.1% trifluoroacetic acid over 20 min:retention time, % area at 254 nm): 359.1 (M + H) 12.1 min, 100%; ChiralHPLC analysis (Chiralcel AD-H, 15% isopropanol in hexanes over 25 mins:retention time, % area at 254 nm): 8.4 min, 99.2%, 11.5 min, 0.8% (98.4de). 110

(S)-1-((R)-3-(3,6- difluoro-9H- carbazol-9-yl)-2- hydroxypropyl)-5-methylpyrrolidin-2- one (300 MHz, CDCl₃): δ 7.68-7.65 (dd, 2H, J = 2.3,8.6 Hz), 7.38-7.34 (dd, 2H, J = 3.9, 8.7 Hz), 7.25-7.18 (td, 2H, J =2.5, 9.0 Hz), 4.73-4.72 (d, 1H, J = 3.0 Hz), 4.38-4.28 (m, 3H),3.47-3.35 (m, 2H), 3.10-3.05 (m, 1H), 2.42-2.27 (m, 2H), 2.20-2.10 (m,1H), 1.57-1.52 (m, 1H), 0.83- 0.81 (d, 3H, J = 6.3 Hz); HPLC analysis:(C18, 5-95% acetonitrile in water + 0.1% trifluoroacetic acid 359.6 (M +H) over 20 min: retention time, % area at 254 nm): 11.5 min, 100%;Chiral HPLC analysis (Chiralcel AD-H, 15% isopropanol in hexanes over 25mins: retention time, % area at 254 nm): 8.4 min, 99.2%, 11.5 min, 0.8%(98.4% de). 111

(R)-1-((R)-3-(9H- carbazol-9-yl)-2- hydroxypropyl)-5-methylpyrrolidin-2- one (300 MHz, CDCl₃): δ 8.10-8.07 (d, 2H, J = 8.4Hz), 7.46-7.44 (m, 4H), 7.27-7.22 (m, 2H), 4.43-4.32 (m, 3H), 4.04-4.03(d, 1H, J = 3.3 Hz), 3.74-3.69 (dd, 1H, J = 7.2, 14.7 Hz), 3.60-3.51(sext, 1H, J = 6.3 Hz), 3.08-3.03 (m, 1H), 2.44-2.36 (m, 2H), 2.24-2.00(m,1H), 1.63-1.53 (m, 1H), 0.97-0.96 (d, 3H, J = 5.7 323.1 (M + H) Hz);HPLC analysis: (C18, 5-95% acetonitrile in water + 0.1% trifluoroaceticacid over 20 min: retention time, % area at 254 nm): 11.4 min, 99.5%;Chiral HPLC analysis (Phenomenex Lux 3μ cellulose-2, 8% isopropanol inhexanes over 60 mins: retention time, % area at 254 nm): 42.0 min, 4.9%,47.8 min, 95.0% (90.0% de). 112

(S)-1-((R)-3-(9H- carbazol-9-yl)-2- hydroxypropyl)-5-methylpyrrolidin-2- one (300 MHz, CDCl₃): δ 8.10-8.07 (d, 2H, J = 8.4Hz), 7.46-7.44 (m, 4H), 7.27-7.22 (m, 2H), 4.73-4.72 (d, 1H, J = 2.4Hz), 4.43-4.28 (m, 3H), 3.44- 3.34 (m, 2H), 3.12-3.07 (m, 1H), 2.45-2.27(m, 2H), 2.17-2.07 (m, 1H), 1.57-1.48 (m, 1H), 0.76-0.74 (d, 3H, J = 6.0Hz); HPLC analysis: (C18, 5-95% acetonitrile in water + 323.1 (M + H)0.1% trifluoroacetic acid over 20 min: retention time, % area at 254nm): 11.4 min, 100%; Chiral HPLC analysis (Phenomenex Lux 3μcellulose-2, 8% isopropanol in hexanes over 60 mins: retention time, %area at 254 nm): 40.0 min, 1.1%, 44.1 min, 97.6% (97.8% de). 113

(R)-1-((S)-3-(9H- carbazol-9-yl)-2- hydroxropyl)-5- methylpyrrolidin-2-one (300 MHz, CDCl₃): δ 8.10-8.07 (d, 2H, J = 8.4 Hz), 7.46-7.44 (m,4H), 7.27-7.22 (m, 2H), 4.73-4.72 (d, 1H, J = 3.0 Hz), 4.43-4.28 (m,3H), 3.44- 3.34 (m, 2H), 3.12-3.07 (m, 1H), 2.45-2.27 (m, 2H), 2.17-2.07(m, 1H), 1.57-1.48 (m, 1H), 0.76-0.74 (d, 3H, J = 6.0 Hz); HPLCanalysis: (C18, 5-95% acetonitrile in water + 323.1 (M + H) 0.1%trifluoroacetic acid over 20 min: retention time, % area at 254 nm):11.5 min, 100%; Chiral HPLC analysis (Phenomenex Lux 3μ cellulose-2, 8%isopropanol in hexanes over 60 mins: retention time, % area at 254 nm):42.6 min, 98.0%, 48.3 min, 1.9% (96.1% de). 114

(R)-1-(3-(3,6- difluoro-9H- carbazol-9-yl)-2- hydroxypropyl)imidazolidin-2- one (300 MHz, CDCl₃): δ 7.68-7.65 (dd, 2H, J = 1.8, 9.0Hz), 7.40-7.35 (dd, 2H, J = 4.1, 9.0 Hz), 7.25-7.17 (td, 2H, J = 2.7,9.0 Hz), 4.43-4.30 (m, 4H), 3.43-3.14 (m, 6H); HPLC analysis: (C18,5-95% acetonitrile in water + 0.1% trifluoroacetic acid over 20 min:retention time, % area at 254 nm): 10.8 min, 100%; Chiral HPLC analysis(Chiralcel AD-H, 30% ethanol in hexanes over 30 346.1 (M + H) mins:retention time, % area at 254 nm): 13.0 min, 99.2%; 21.6 min, 0.7% (98%ee). 115

(S)-1-((R)-3-(3,6- difluoro-9H- carbazol-9-yl)-2- hydroxypropyl)-3-methylpyrrolidin-2- one (300 MHz, CDCl₃): δ 7.69 (dd, 2H, J = 9.0, 2.4Hz), 7.37 (dd, 2H, J = 9.0, 4.2 Hz), 7.23 (td, 2H, J = 9.0, 2.4 Hz),4.45-4.25 (m, 3H), 3.96 (d, 1H, J = 4.2 Hz), 3.48 (dd, 1H, J = 14.4, 6.6Hz), 3.38-3.15 (m, 3H), 2.54 (m, 1H), 2.25 (m, 1H), 1.66 (m, 1H), 1.23(d, 3H, J = 6.9 Hz); HPLC analysis: (C18, 10-90% acetonitrile in waterover 20 min: retention time, % area at 254 nm): 12.2 min, 100%; 359.1(M + H) Chiral HPLC analysis (Phenomenex Lux 3μ cellulose-2, 15%isopropanol in hexanes over 30 mins: retention time, % area at 254 nm):14.6 min, 96.9% de, 100% ee. 116

(R)-1-((R)-3-(3,6- difluoro-9H- carbazol-9-yl)-2- hydroxypropyl)-3-methylpyrrolidin-2- one (300 MHz, CDCl₃): δ 7.69 (dd, 2H, J = 9.0, 2.4Hz), 7.37 (dd, 2H, J = 9.0, 4.2 Hz), 7.23 (td, 2H, J = 9.0, 2.4 Hz),4.45-4.25 (m, 3H), 4.01 (d, 1H, J = 2.7 Hz), 3.53 (dd, 1H, J = 14.1, 6.9Hz), 3.40-3.15 (m, 3H), 2.52 (m, 1H), 2.24 (m, 1H), 1.68 (m, 1H), 1.23(d, 3H, J = 7.2 Hz); HPLC analysis: (C18, 10-90% acetonitrile in waterover 20 min: retention time, % area at 254 nm): 12.2 min, 100%; 359.2(M + H) Chiral HPLC analysis (Phenomenex Lux 3μ cellulose-2, 15%isopropanol in hexanes over 30 mins: retention time, % area at 254 nm):12.3 min, 100% de, 100% ee. 117

(S)-14(R)-3-(9H- carbazol-9-yl)-2- hydroxypropyl)-3- methylpyrrolidin-2-one (300 MHz, CDCl₃): δ 8.11 (d, 2H, J = 7.8 Hz), 7.53-7.42 (m, 4H),7.32- 7.22 (m, 2H), 4.50-4.30 (m, 3H), 4.01 (s, 1H), 3.51 (dd, 1H, J =14.4, 6.9 Hz), 3.35-3.20 (m, 2H), 3.14 (td, 1H, J = 9.3, 3.3 Hz), 2.54(m, 1H), 2.22 (m, 1H), 1.64 (m, 1H), 1.23 (d, 3H, J = 7.8 Hz); HPLCanalysis: (C18, 10-90% acetonitrile in water 323.2 (M + H) over 20 min:retention time, % area at 254 nm): 11.6 min, 99.2%; Chiral HPLC analysis(Phenomenex Lux 3μ cellulose-2, 15% isopropanol in hexanes over 30 mins:retention time, % area at 254 nm): 13.6 min, 100% de, 100% ee. 118

(R)-1-((R)-3-(9H- carbazol-9-yl)-2- hydroxypropyl)-3-methylpyrrolidin-2- one (300 MHz, CDCl₃): δ 8.11 (d, 2H, J = 7.8 Hz),7.53-7.42 (m, 4H), 7.32- 7.22 (m, 2H), 4.50-4.30 (m, 3H), 4.07 (s, 1H),3.55 (dd, 1H, J = 14.1, 6.9 Hz), 3.30-3.15 (m, 3H), 2.51 (m, 1H), 2.20(m, 1H), 1.64 (m, 1H), 1.22 (d, 3H, J = 7.5 Hz); HPLC analysis: (C18,10-90% acetonitrile in water over 20 min: retention time, 323.1 (M + H)% area at 254 nm): 11.6 min, 100%; Chiral HPLC analysis (Phenomenex Lux3μ cellulose-2, 15% isopropanol in hexanes over 30 mins: retention time,% area at 254 nm): 13.6 min, 100% de, 100% ee. 119

(R)-1-((R)-3-(3,6- difluoro-9H- carbazol-9-yl)-2- hydroxypropyl)-3-fluoropyrrolidin-2- one (300 MHz, CDCl₃): δ 7.69-7.65 (dd, 2H, J = 2.4,8.9 Hz), 7.37-7.33 (dd, 2H, J = 8.9, 4.5Hz), 7.24-7.18 (td, 2H, J = 9.0,2.7 Hz), 5.20-4.98 (ddd, 1H, J = 6.0, 7.5, 52 Hz), 4.45-4.33 (m, 3H),3.59-3.32 (m, 4H), 3.07- 3.06 (d, 1H, J = 4.2 Hz), 2.60-2.10 (m, 2H);HPLC analysis: (C18, 10- 90% acetonitrile in water over 20 min:retention time, % area at 254 nm): 11.6 min, 100%; Chiral HPLC 363.3(M + H) analysis (Phenomenex Lux 3μ cellulose-2, 15% isopropanol inhexanes over 60 mins: retention time, % area at 254 nm): 33.0 min,98.3%, 47.9 min, 1.6% (96.6% de). 120

(S)-1-((R)-3-(3,6- difluoro-9H- carbazol-9-yl)-2- hydroxypropyl)-3-fluoropyrrolidin-2- one (300 MHz, CDCl₃): δ 7.68-7.65 (dd, 2H, J = 2.7,8.4 Hz), 7.37-7.33 (dd, 2H, J = 4.1, 9.5 Hz), 7.24-7.18 (td, 2H, J =8.9, 2.5 Hz), 5.17-4.94 (ddd, 1H, J = 6.0, 7.7, 53 Hz), 4.38-4.30 (m,3H), 3.52-3.30 (m, 5H), 2.50- 2.11 (m, 2H); HPLC analysis: (C18, 10-90%acetonitrile in water over 20 min: retention time, % area at 254 nm):11.6 min, 98.3%; Chiral HPLC analysis (Chiralcel AD-H, 30% 363.4 (M + H)ethanol in hexanes over 30 mins: retention time, % area at 254 nm): 7.3min, 100% (100% de). 121

(R)-1-((R)-3-(9H- carbazol-9-yl)-2- hydroxypropyl)-3-fluoropyrrolidin-2- one (300 MHz, CDCl₃): δ 8.11-8.08 (d, 2H, J = 7.8Hz), 7.50-7.42 (m, 4H), 7.28-7.23 (m, 2H), 5.19-4.97 (ddd, 1H, J = 52.8,6.3, 7.5 Hz), 4.48-4.34 (m, 3H), 3.55-3.12 (m, 5H), 2.47- 2.13 (m, 2H);HPLC analysis: (C18, 10-90% acetonitrile in water + 0.1% trifluoroaceticacid over 20 min: retention time, % area at 254 nm): NA 11.0 min, 100%;Chiral HPLC analysis (Phenomenex Lux 3μ cellulose-2, 15% isopropanol inhexanes over 60 mins: retention time, % area at 254 nm): 34.4 min,98.9%, 46.8 min, 1.0% (97.9% de). 122

(S)-14(R)-3-(9H- carbazol-9-yl)-2- hydroxypropyl)-3- fluoropyrrolidin-2-one (300 MHz, CDCl₃): δ 8.09-8.06 (d, 2H, J = 8.1 Hz), 7.49-7.41 (m,4H), 7.27-7.21 (m, 2H), 5.12-4.90 (ddd, 1H, J = 52.2, 6.0, 8.1 Hz),4.39-4.32 (m, 3H), 3.48-3.25 (m, 5H), 2.45- 2.33 (m, 1H), 2.22-2.06 (m,1H); HPLC analysis: (C18, 10-90% acetonitrile + 0.1% trifluoroaceticacid in water over 20 min: retention NA time, % area at 254 nm): 11.0min, 100%; Chiral HPLC analysis (Chiralcel AD-H, 15% ethanol in hexanesover 30 mins: retention time, % area at 254 nm): 19.5 min, 100% (100%de). 123

(R)-1-((S)-3-(9H- carbazol-9-yl)-2- hydroxy-2- methylpropyl)-3-fluoropyrrolidin-2- one (300 MHz, CDCl₃): δ 8.10-8.07 (m, 2H), 7.50-7.42(m, 4H), 7.28-7.23 (m, 2H), 5.20-4.99 (ddd, 1H, J = 5.9, 7.4, 52.2 Hz),4.32 (s, 2H), 3.78-3.46 (m, 4H), 2.51 (s, 1H), 2.49-2.18 (m, 2H), 1.30(s, 3H); HPLC analysis: (C18, 10-90% acetonitrile in water over 20 min:retention time, % area at 254 nm): 11.6 min, 100%; Chiral 341.1 (M + H)HPLC analysis (Chiralcel AD-H, 15% isopropanol in hexanes over 60 mins:retention time, % area at 254 nm): 22.0 min, 100% (100% de). 124

(S)-1-((S)-3-(9H- carbazol-9-yl)-2- hydroxy-2- methylpropyl)-3-fluoropyrrolidin-2- one (300 MHz, CDCl₃): δ 8.10-8.07 (m, 2H), 7.50-7.42(m, 4H), 7.28-7.23 (m, 2H), 5.20-4.99 (dt, 1H, J = 7.1, 52.8 Hz),4.40-4.29 (m, 2H), 3.74- 3.48 (m, 4H), 2.62 (s, 1H), 2.53-2.18 (m, 2H),1.28 (s, 3H); HPLC analysis: (C18, 10-90% acetonitrile in water over 20min: retention time, % area at 254 nm): 11.6 min, 100%; 341.1 (M + H)Chiral HPLC analysis (Chiralcel AD-H, 30% ethanol in hexanes over 60mins: retention time, % area at 254 nm): 8.3 min, 100% (100% de). 125

(S)-1-((S)-3-(3,6- difluoro-9H- carbazol-9-yl)-2- hydroxy-2-methylpropyl)-3- fluoropyrrolidin-2- one (300 MHz, CDCl₃): δ 7.68-7.64(dd, 2H, J = 2.7, 8.4 Hz), 7.42-7.38 (dd, 2H, J = 4.2, 8.7 Hz),7.23-7.16 (td, 2H, J = 2.6, 9.3 Hz), 5.23-5.01 (ddd, 1H, J = 52.8, 5.7,7.5 Hz), 4.35-4.24 (m, 2H), 3.80-3.73 (m, 1H), 3.59- 3.46 (m, 3H),2.66-2.19 (m, 2H), 1.26 (s, 3H); HPLC analysis: (C18, 10-90%acetonitrile in water + 0.1% trifluoroacetic acid over 20 min: retentiontime, % area at 254 nm): NA 12.2 min, 100%; Chiral HPLC analysis(Phenomenex Lux 3μ cellulose-2, 4% ethanol + 4% methanol in hexanes over45 mins: retention time, % area at 254 nm): 26.5 min, 97.6%, 30.9 min,0.4%, 36.5 min, 2.0% (95.5% de, 97.1% ee). 126

(R)-1-((S)-3-(3,6- difluoro-9H- carbazol-9-yl)-2- hydroxy-2-methylpropyl)-3- fluoropyrrolidin-2- one (300 MHz, CDCl₃): δ 7.69-7.65(dd, 2H, J = 2.7, 8.7 Hz), 7.43-7.39 (dd, 2H, J = 4.2, 8.7 Hz),7.24-7.20 (td, 2H, J = 2.6, 8.7 Hz), ), 5.24-4.97 (ddd, 1H, J = 52.8,5.4, 7.5 Hz), 4.28 (s, 2H), 3.77-3.71 (m, 1H), 3.60-3.46 (m, 3H), -3.12(m, 5H), 2.54 (s, 1H), 2.53-2.22 (m, 2H), 1.28 (s, 3H); HPLC analysis:(C18, 10-90% acetonitrile in water + 0.1% trifluoroacetic acid over 20min: 377.0 (M + H) retention time, % area at 254 nm): 12.2 min, 100%;Chiral HPLC analysis (Phenomenex Lux 3μ cellulose-2, 4% ethanol + 4%methanol in hexanes over 45 mins: retention time, % area at 254 nm):26.5 min, 3.1%, 36.6 min, 96.9% (93.7% de). 127

(R)-1-((R)-3-(9H- carbazol-9-yl)-2- hydroxy-2- methylpropyl)-3-fluoropyrrolidin-2- one (300 MHz, CDCl₃): δ 8.11-8.08 (d, 2H, J = 7.8Hz), 7.52-7.43 (m, 4H), 7.28-7.23 (m, 2H), 5.24-5.02 (ddd, 1H, J = 52.8,6.2, 7.5 Hz), 4.48-4.31 (m, 3H), 3.75-3.50 (m, 4H), 2.57 (s, 1H),2.54-2.23 (m, 2H), 1.29 (s, 3H); HPLC analysis: (C18, 10-90%acetonitrile in water + 0.1% trifluoroacetic acid over 20 min: 341.0(M + H) retention time, % area at 254 nm): 11.6 min, 100%; Chiral HPLCanalysis (Chiralcel AD-H, 30% isopropanol in hexanes over 30 mins:retention time, % area at 254 nm): 9.6 min, 0.8%, 15.6 min, 98.7%, 22.0min, 0.5% (97.3% de). 128

(S)-1-((R)-3-(9H- carbazol-9-yl)-2- hydroxy-2- methylpropyl)-3-fluoropyrrolidin-2- one (300 MHz, CDCl₃): δ 8.11-8.08 (d, 2H, J = 7.4Hz), 7.51-7.43 (m, 4H), 7.28-7.22 (m, 2H), 5.23-5.02 (ddd, 1H, J = 52.2,5.1, 7.2 Hz), 4.34 (s, 2H), 3.79-3.46 (m, 4H), 2.55-2.20 (m, 2H), 2.44(s, 1H), 1.31 (s, 3H); HPLC analysis: (C18, 10-90% acetonitrile inwater + 0.1% trifluoroacetic acid over 20 min: 341.1 (M + H) retentiontime, % area at 254 nm): 11.7 min, 100%; Chiral HPLC analysis (ChiralcelAD-H, 30% ethanol in hexanes over 30 mins: retention time, % area at 254nm): 9.6 min, 99.6%, 15.9 min, 0.4% (99.2% de). 129

(S)-1-((R)-3-(9H- carbazol-9-yl)-2- hydroxypropyl)-5- methylpiperidin-2-one (300 MHz, CDCl₃): δ 8.10 (d, 2H, J = 8.4 Hz), 7.50-7.40 (m, 4H),7.30- 7.20 (m, 2H), 4.50-4.30 (m, 3H), 4.25 (d, 1H, J = 3.6 Hz), 3.90(dd, 1H, J = 14.1, 8.1 Hz), 3.09 (dd, 1H, J = 14.1, 2.1 Hz), 2.98 (ddd,1H, J = 11.7, 5.1, 1.8 Hz), 2.83 (t, 1H, J = 10.8 Hz), 2.49 (ddd, 1H, J= 18.0, 6.0, 3.0 Hz), 2.36 (ddd, 1H, J = 18.0, 11.1, 6.3 Hz), 1.95-1.75(m, 2H), 337.4 (M + H) 1.43 (m, 1H), 0.88 (d, 3H, J = 6.6 Hz); ChiralHPLC analysis (Chiralcel AD-H, 8% ethanol in hexanes over 45 mins:retention time, % area at 254 nm): 25.7 min, 98.6% de, 100% ee. 130

(S)-1-((R)-3-(3,6- difluoro-9H- carbazol-9-yl)-2- hydroxypropyl)-5-methylpiperidin-2- one (300 MHz, CDCl₃): δ 7.69 (dd, 2H, J = 8.7, 2.4Hz), 7.39 (dd, 2H, J = 9.0, 4.2 Hz), 7.23 (td, 2H, J = 8.7, 2.4 Hz),4.45-4.25 (m, 3H), 4.18 (d, 1H, J = 3.3 Hz), 3.84 (dd, 1H, J = 14.1, 8.1Hz), 3.12 (dd, 1H, J = 14.1, 2.4 Hz), 3.07 (m, 1H), 2.89 (t, 1H, J =11.1 Hz), 2.50 (ddd, 1H, J = 18.0, 6.3, 3.3 Hz), 2.37 (ddd, 1H, J =18.0, 11.1, 6.3 Hz), 2.00-1.77 (m, 2H), 1.45 (m, 1H), 0.94 (d, 3H, J =6.6 Hz); Chiral HPLC analysis 373.5 (M + H) (Chiralcel AD-H, 15% ethanolin hexanes over 40 mins: retention time, % area at 254 nm): 12.2 min,100% de, 100% ee. 131

(R)-1-((R)-3-(9H- carbazol-9-yl)-2- hydroxypropyl)-5- methylpiperidin-2-one (300 MHz, CDCl₃): δ 8.11 (d, 2H, J = 7.8 Hz), 7.50-7.40 (m, 4H),7.30- 7.20 (m, 2H), 4.66 (s, 1H), 4.50-4.30 (m, 3H), 4.00 (dd, 1H, J =14.1, 8.7 Hz), 3.00 (dd, 1H, J = 12.0, 5.4 Hz), 2.90 (d, 1H, J = 14.1Hz), 2.73 (t, 1H, J = 11.7 Hz), 2.49 (ddd, 1H, J = 18.0, 6.0, 3.0 Hz),2.37 (ddd, 1H, J = 18.0, 11.1, 6.0 Hz), 1.95-1.70 (m, 2H), 1.38 (m, 1H),0.88 (d, 3H, J = 338.0 (M + H) 6.6 Hz); Chiral HPLC analysis (ChiralcelAD-H, 8% ethanol in hexanes over 45 mins: retention time, % area at 254nm): 37.2 min, 100% de, 100% ee. 132

(S)-1-((S)-3-(9H- carbazol-9-yl)-2- hydroxypropyl)-5- methylpiperidin-2-one (300 MHz, CDCl₃): δ 8.11 (d, 2H, J = 7.8 Hz), 7.55-7.43 (m, 4H),7.32- 7.22 (m, 2H), 4.64 (s, 1H), 4.50-4.30 (m, 3H), 4.00 (dd, 1H, J =14.1, 8.7 Hz), 3.00 (ddd, 1H, J = 12.3, 4.8, 1.8 Hz), 2.90 (d, 1H, J =14.4 Hz), 2.73 (t, 1H, J = 11.1 Hz), 2.55-2.30 (m, 2H), 1.98-1.72 (m,2H), 1.37 (m, 1H), 0.89 (d, 3H, J = 6.6 Hz); Chiral HPLC analysis(Chiralcel AD-H, 8% 337.9 (M + H) ethanol in hexanes over 45 mins:retention time, % area at 254 nm): 20.3 min, 100% de, 100% ee. 133

(R)-1-((S)-3-(9H- carbazol-9-yl)-2- hydroxypropyl)-5- methylpiperidin-2-one (300 MHz, CDCl₃): δ 8.11 (d, 2H, J = 7.8 Hz), 7.50-7.40 (m, 4H),7.30- 7.20 (m, 2H), 4.50-4.30 (m, 3H), 4.23 (d, 1H, J = 2.7 Hz), 3.90(dd, 1H, J = 14.1, 7.8 Hz), 3.10 (dd, 1H, J = 14.1, 2.1 Hz), 2.99 (ddd,1H, J = 11.7, 4.5, 1.8 Hz), 2.84 (t, 1H, J = 10.8 Hz), 2.49 (ddd, 1H, J= 18.0, 6.0, 3.0 Hz), 2.36 (ddd, 1H, J = 18.0, 11.1, 6.6 Hz), 1.95-1.75(m, 2H), 337.9 (M + H) 1.43 (m, 1H), 0.90 (d, 3H, J = 6.6 Hz); ChiralHPLC analysis (Chiralcel AD-H, 8% ethanol in hexanes over 45 mins:retention time, % area at 254 nm): 21.6 min, 97.8% de, 100% ee. 134

(R)-1-((R)-3-(3,6- difluoro-9H- carbazol-9-yl)-2- hydroxypropyl)-5-methylpiperidin-2- one (300 MHz, CDCl₃): δ 7.69 (dd, 2H, J = 9.0, 2.4Hz), 7.39 (dd, 2H, J = 9.3, 4.2 Hz), 7.23 (td, 2H, J = 9.3, 2.7 Hz),4.56 (s, 1H), 4.45-4.25 (m, 3H), 3.94 (dd, 1H, J = 14.1, 9.0 Hz), 3.06(dd, 1H, J = 12.0, 5.1 Hz), 2.90 (d, 1H, J = 14.1 Hz), 2.80 (t, 1H, J =11.4 Hz), 2.55-2.30 (m, 2H), 2.00- 1.75 (m, 2H), 1.40 (m, 1H), 0.93 (d,3H, J = 7.2 Hz); Chiral HPLC analysis (Chiralcel AD-H, 15% ethanol inhexanes over 40 mins: 373.9 (M + H) retention time, % area at 254 nm):15.7 min, 100% de, 100% ee. 135

(S)-1-((R)-3-(3,6- difluoro-9H- carbazol-9-yl)-2- hydroxypropyl)-4-methylimidazolidin- 2-one (300 MHz, CDCl₃): δ 7.68 (dd, 2H, J = 9.0, 2.4Hz), 7.39 (dd, 2H, J = 9.0, 4.2 Hz), 7.23 (td, 2H, J = 9.0, 2.7 Hz),4.52 (s, 1H), 4.45-4.26 (m, 3H), 4.21 (brs, 1H), 3.83 (m, 1H), 3.55 (t,1H, J = 8.7 Hz), 3.39 (dd, 1H, J = 14.7, 7.2 Hz), 3.09 (dd, 1H, J =15.0, 2.4 Hz), 2.96 (dd 1H, J = 9.0. 6.9 Hz), 1.24 (d, 3H, J = 5.7 Hz);Chiral HPLC analysis (Chiralcel AD-H, 15% ethanol in hexanes over 60360.2 (M + H) mins: retention time, % area at 254 nm): 29.1 min, 100%de, 100% ee. 136

(R)-1-((R)-3-(3,6- difluoro-9H- carbazol-9-yl)-2- hydroxypropyl)-4-methylimidazolidin- 2-one (300 MHz, CDCl₃): δ 7.68 (dd, 2H, J = 9.0, 2.4Hz), 7.39 (dd, 2H, J = 9.0, 4.2 Hz), 7.22 (td, 2H, J = 9.0, 2.7 Hz),4.66 (s, 1H), 4.50-4.20 (m, 4H), 3.81 (m, 1H), 3.38 (t, 1H, J = 8.7 Hz),3.22 (s, 2H), 2.99 (dd, 1H, J = 8.4, 6.6 Hz), 1.24 (d, 3H, J = 5.7 Hz);Chiral HPLC analysis (Chiralcel AD-H, 15% ethanol in hexanes over 60mins: retention time, % area at 254 nm): 27.0 min, 100% de, 100% ee.360.2 (M + H) 137

(S)-1-((R)-3-(9H- carbazol-9-yl)-2- hydroxypropyl)-4-methylimidazolidin- 2-one (300 MHz, CDCl₃): δ 7.68 (dd, 2H, J = 9.0, 2.4Hz), 7.39 (dd, 2H, J = 9.0, 4.2 Hz), 7.22 (td, 2H, J = 9.0, 2.7 Hz),4.66 (s, 1H), 4.50-4.20 (m, 4H), 3.81 (m, 1H), 3.38 (t, 1H, J = 8.7 Hz),3.22 (s, 2H), 2.99 (dd, 1H, J = 8.4, 6.6 Hz), 1.24 (d, 3H, J = 5.7 Hz);Chiral HPLC analysis (Chiralcel AD-H, 15% ethanol in 324.3 (M + H)hexanes over 60 mins: retention time, % area at 254 nm): 28.8 min, 100%de, 100% ee. 138

(R)-1-((R)-3-(9H- carbazol-9-yl)-2- hydroxypropyl)-4-methylimidazolidin- 2-one (300 MHz, CDCl₃): δ 8.11 (d, 2H, J = 7.8 Hz),7.55-7.42 (m, 4H), 7.35- 7.20 (m, 2H), 4.53 (brs, 1H), 4.48- 4.30 (m,4H), 3.80 (m, 1H), 3.34 (t, 1H, J = 8.7 Hz), 3.28-3.15 (m, 2H), 2.95(dd, 1H, J = 8.7, 6.9 Hz), 1.23 (d, 3H, J = 6.6 Hz); Chiral HPLCanalysis (Chiralcel AD-H, 15% ethanol in hexanes over 60 mins: 324.2(M + H) retention time, % area at 254 nm): 25.1 min, 100% de, 100% ee.

Specific assays useful for evaluating the compounds of formula I includethe Per2 Assay for Evaluating the Potency of Test Compounds and the Cry1Assay for Evaluating the Target of Test Compounds, as described below.

Example 3 Per2 Assay for Evaluating the Potency of Test Compounds

Compounds were screened by using a high-throughput circadian assaysystem as previously described in Zhang, E. E. et al. Cell, 2009, 139,199-210. In brief, stable U2OS reporter cells harboring Per2-dLuc wereplated at a density of 30,000 cells/well in Corning 96-well, solidwhite, flat bottom, TC-treated microplates (Corning®), and incubated for48 hours at 37° C. in the presence of 5% CO₂ in a medium of Dulbecco'sModified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum(FBS) and penicillin (100 units/mL)-streptomycin (100 μg/mL). Compoundsof formula I are solubilized in dimethylsulfoxide (DMSO), typically at aconcentration of 2 mg/mL. DMSO stocks are then serially diluted in DMSO,typically diluting 3-fold for each dilution step. Following the 48 hperiod, cell culture medium is removed from plated cells and cells aresynchronized with 200 μL/well of complete cell culture medium (describedabove), supplemented with 5 μM forskolin (Tocris®) and 1 mM beetleluciferin (Promega®). Immediately following synchronization, 1 μL ofcompound dilution is added to each well. Plates are sealed, shakenbriefly, and gene expression was monitored by measuring luminescence(Tecan® Infinite M200 or Tecan® Infinite M200 Pro) continuously for aminimum of 3 days at 35° C. Raw luminescence data (counts) are firstanalyzed using Multicycle™ software (Actimetrics, Inc.) to determine theamplitude (amp), period, and phase (phz) for each compoundconcentration. The period length for control wells (i.e. no compound,DMSO only) should be 26-30 h. Amp data are then plotted against thelogarithm compound concentration (M) and analyzed by nonlinearregression analysis to determine the EC₅₀.

The following table provides Per2 EC₅₀ data for the specified compounds.The EC_(50s) are reported as micromolar concentration.

TABLE 1 Per2 assay data Compound Per2 EC₅₀ (μM) 1 0.171 2 0.736 3 0.2994 0.192 5 0.536 6 0.719 7 0.625 8 0.527 9 0.300 10 0.487 11 0.120 120.909 13 0.288 14 0.541 15 0.164 16 0.463 17 0.417 18 0.338 19 0.547 200.379 21 1.042 22 0.261 23 0.399 24 0.332 25 0.079 26 1.051 27 0.478 280.824 29 0.422 30 0.895 31 0.832 32 0.539 33 0.625 34 0.286 35 0.678 360.760 37 0.224 38 1.216 39 0.342 40 0.484 41 0.373 42 0.420 43 1.033 441.060 45 0.052 46 0.618 47 0.299 48 0.120 49 0.137 50 0.204 51 0.265 521.355 53 1.337 54 0.808 55 0.738 56 1.057 57 0.329 58 0.716 59 0.986 600.331 61 0.710 62 0.370 63 0.541 64 0.462 65 1.030 66 0.523 67 0.225 680.288 69 0.137 70 0.061 71 0.188 72 0.389 73 1.111 74 0.284 75 0.171 760.609 77 0.620 78 0.440 79 0.923 80 1.105 81 0.816 82 1.192 83 0.147 840.325 85 0.667 86 0.497 87 0.317 88 0.310 89 0.438 90 0.605 91 0.621 920.527 93 0.129 94 0.833 95 0.568 96 0.493 97 0.315 98 0.269 99 0.555 1000.721 101 0.417 102 0.455 103 0.940 104 0.115 105 0.054 106 0.220 1070.089 108 0.750 109 0.226 110 0.830 111 0.095 112 0.470 113 0.948 1140.287 115 0.500 116 0.311 117 0.188 118 0.120 119 0.184 120 0.302 1210.210 122 0.263 123 0.311 124 0.309 125 0.451 126 0.958 127 0.535 1280.172 129 0.748 130 0.895 131 0.280 132 1.133 133 0.838 134 0.397 1351.129 136 0.826 137 0.766 138 0.520

One of ordinary skill in the art could readily optimize this assay todetermine Per2 EC₅₀ data for any of the compounds described herein.

Example 4 Thermal Shift Binding Assay

Binding of compounds to the isolated FAD-binding domain of human CRY1protein (hCRY1) was determined using a differential scanning fluorimetry(‘thermal shift’) assay (Pantoliano et al. (2001) J Biomol Screening 6,429; Niesen et al. (2007) Nature Protocols 2, 2212). The FAD-bindingdomain of hCRY1 (amino acid residues 1-494) with a C-terminal Myc-DDKtag (FADBD), was produced by transient transfection of HEK293T cells(Catalog # CRL-3216, American Type Culture Collection) and purified byanti-FLAG affinity chromatography (Catalog # A2220, Sigma-Aldrich).FADBD (0.5 μg per well) was incubated with dilutions of compounds inDMSO (5% DMSO final concentration in reaction) in 17.5 μl Tris-bufferedsaline (TBS) for 10 minutes on ice, then 2.5 μl of 8× Sypro-Orange Dye(Life Technologies) was added to each well. Triplicate wells wereassayed for each compound concentration. The melting temperatures weremeasured in an ABI7500 quantitative PCR instrument using the melt curvemode with a thermal profile of 2 minutes at 25° C., followed by a ramprate of 1° C./minute up to 99° C. The melting temperature for each wellwas determined from the first derivative of the melt curve. The changein melting temperature (ΔTm) was obtained by subtraction of the meltingtemperature of the FADBD in 5% DMSO alone. As shown in FIG. 21, adose-dependent increase in ΔTm was observed for Compound 72, indicatingthat the compound physically associates with the hCRY1 FADBD protein.

Example 5 In Vivo Effect on Clock Gene and Gluconeogenic Gene Expression

In this example, the effect of carbazole-containing amids, carbamates,and ureas on clock and gluconeogenic gene expression in various mousemodels were examined. Specifically, four different mouse models wereused: ICR mice, Balb/c mice, C57Bl/6J DIO (diet-induced obesity) mice,and db/db mice. Both the C57Bl/6J DIO and db/db mice are art-recognizedmodels of diabetes, obesity, and dyslipidemia. Diet induced obese (DIO)mice, which exhibit a type II diabetic phenotype in response to high fatfeeding develop obesity, hyperinsulinemia, insulin resistance andglucose intolerance (Srinivasan and Ramarao, 2007). The db/db mouse(lepr^(db) mouse) has a mutation in the db gene, which encodes theleptin receptor. Db/db mice are spontaneously hyperphagic and becomeobese, hyperglycemic, hyperinsulinemic and insulin resistant.

The in vivo studies examining the effect of Cry modulator compounds ongene expression were performed using the various experimental methodsdescribed below.

Four Day Study.

Male ICR mice (weighing 30-35 g), obtained from Charles RiverLaboratories (Hollister, Calif.), were used for the experiment followingat least 3 days of acclimation. Mice were dosed with vehicle (WFI or 10%Kolliphor) or compound (50 mg/kg, dose volume 5 mL/kg, PO) for 4 daysBID (twice a day), beginning in the afternoon of the first day. The lastdose of compound or vehicle was administered on the morning of harvest.On the day of harvest (6 hours following the final dose), mice wereeuthanized by CO₂ asphyxiation and 50 mg of liver tissue was excised andplaced in a tube containing 500 μl of RNA later.

24 Hour Gene Expression Studies.

Male C57Bl/6J DIO mice were purchased at 17 weeks of age (The JacksonLaboratory, Sacramento, Calif.) and acclimated for a 2 week periodbefore being used for the experiment. Overall group size was 15 pertreatment, divided into 5 time-points to give a final group size of 3mice. Mice were dosed with vehicle (water for injection (WFI) or the CryModulator compound Compound 72 (50 mg/kg in WFI) at a dose volume of 5ml/kg, BID, via oral gavage for 2 days, with a final fifth doseadministered 12 hours prior to harvest.

In total, each mouse received 5 doses of compound over the course of theexperiment. Mice were weighed and randomly assigned to each treatmentbased on weight the evening before the commencement of the study.Beginning on Day 3 at 3:00 PM, a group of animals from the vehicle andCompound 72-treated groups were euthanized using CO₂ asphyxiation, 50 mgof liver, epididymal fat, and skeletal muscle, respectively, wereexcised and placed in tube containing 500 μl of RNALater. This proceduretook place for the remainder of the time-point groups at their givenharvest times. Plasma samples were also taken for each animal andfrozen, to be used for later measurement of compound levels.

Mouse Liver Total RNA Preparation.

E.Z.N.A.® HP Total RNA Isolation Kits (R6812-01 and the protocoldescribed in the Manual, Revised 2010) were utilized to prepare andisolate RNA from the liver samples. To prepare the RNA samples, 10-30 mgof sample was removed from RNA-Later and placed in a 1.5 ml microfugetube. GTC lysis buffer (700 μl) was added to the tissue, which washomogenized with a rotor-stator homogenizer (e.g. Tissue-Tearor, Model#985370 BioSpec Products with 4.5 mm probe, Cat. #985370-04), and thencentrifuged at full speed (≧13,000×g) for 5 minutes. The clearedsupernatant was transferred by pipetting to a DNA Clearance ColumnPre-inserted in a 2 mL Collection Tube. The assembled column wascentrifuged at 13,000×g for 1 minute, and the flow-through was saved. Anequal volume (700 μl) of 70% ethanol was added to the lysate and mixed.The sample was then applied to a HiBind RNA spin column placed into a 2ml collection tube, which was centrifuged at 10,000×g for 60 seconds atroom temperature. RNA Wash Buffer I (250 μl) was added by pipettingdirectly onto a new HiBind RNA column inserted in a 2 ml collectiontube. The assembled column was centrifuged at 10,000×g for 60 seconds.The RNA column was placed into a new 2 ml collection tube. DNase I stocksolution is pipetted (75 μl) directly onto the surface of the HiBind RNAresin in each column (Using DNAse Digestion with RNase Free DNase Set(E1091): for each HiBind RNA column, the DNase I stock solution wasprepared as followed: E.Z.N.A.® DNase I Digestion Buffer 73.5 μl, RNaseFree DNase I (20 Kunitz/μl) 1.5 μl=Total Volume 75 μl.). The column withbound RNA was incubated at room temperature (25-30° C.) for 15 minutes.RNA Wash Buffer I (500 μl) was added to the column and placed on a benchtop for 2 minutes. After centrifuging at 10,000×g for 60 seconds,discarding the flow-through, 500 μl of RNA Wash Buffer II was added andcentrifuged at 10,000×g for 60 seconds. Another 500 μl of RNA WashBuffer II was added and the column assembly was centrifuged at 10,000×gfor 60 seconds. The column was centrifuged for 2 minutes at maximumspeed to completely dry the HiBind matrix. The column was placed in aclean 1.5 ml microcentrifuge tube and 40-70 μl of molecular biologygrade water is added. After sitting for 1 minute, the column wascentrifuged for 2 minutes at maximum speed to elute the RNA. Theisolated RNA was collected into the collection tube.

Mouse Blood Total RNA Preparation.

For whole blood RNA studies, male db/db mice (9 weeks of age, TheJackson Laboratory, Bar Harbor, Me.) were used for the experiment withn=8 mice for each experimental group. Mice were dosed with Compound 72(100 mg/kg, P.O; dose volume 5 ml/kg, in 10% Kolliphor), or 10%Kolliphor, once daily for three days at ZT0 (7:00 am) (ZT refers toZeitgeber Time, and indicates the time at which the lights were turnedon to stimulate day in the mouse facility). On the final day at ZT7.5(2:30 pm), animals were euthanized using CO₂ asphyxiation, and the bloodcollected from the heart via cardiac puncture. The blood was placed inRNALater solution to 1.5 ml total volume. Total RNA was prepared usingan Ambion Mouse RiboPure Blood RNA Isolation Kit AM1951 as follows.Samples were centrifuged for 3 minutes, discarding the supernatant. Twoml of lysis solution was added and vortexed, transferred to a 15 ml tubefollowed by the addition of microliters 3M Sodium Acetate. Lysis bufferwas added to a total volume of 3.8 ml and vortexed. The sample mixturewas extracted with 1.5 ml Acid Phenol: Chloroform and the aqueous phasewas recovered. After adding 0.5 volumes of 100% ethanol and vortexing,the samples were passed through a filter column provided in the kit andwashed with 750 microliters of wash buffer 1. The filter was washed with2 passes of 750 microliters wash buffer 2/3 and dried. RNA was eluted in200 microliters of molecular biology grade water (RNase-free).

Quantitation of Total RNA.

RediPlate 96 RiboGreen RNA Quantification Kit (Invitrogen) RediPlatestandard curve was prepared by transferring 20 μl reconstituted RNAstandard to a RediPlate well reconstituted in 180 μl RediPlate TEbuffer. For liver RNAs prepared from 30-100 mg of tissue using a kitsimilar to the Omega Bio-Tek HP Total RNA Kit and eluted in RNAse-freewater in a volume of 50 μl (microliters), 5 μl of the total RNA isdiluted in 195 μl of RediPlate TE buffer (1:40 dilution of RNA), mix.After transferring 5 μl to RediPlate well reconstituted in 195 μl TEbuffer and incubating 10 min. at RT, Fluorescence Intensity of wellswith standards and samples were read in a Tecan M200 with Excitation setat 480 nm and Emission set at 520 nm with the gain set to ˜70%.Alternatively, one can use the FlexStation 3 with Ex 488 nm and Em 525nm and a Cutoff of 515 nm. A standard curve was generated in GraphPadPrism and sample readings (unknowns) are interpolated via linearregression analysis, and the RNA sample concentrations were calculated.

Preparation of cDNA.

High-Capacity cDNA Reverse Transcription Kit (Invitrogen) 10×RT Buffer(40-70 μl), dNTPs and random primers were thawed on ice. Using the sameamount of input RNA for each sample (usually 0.5-4.0 μg) reactions wereset up in a total volume of 40 μl. Appropriate amounts of total RNA andNuclease-Free H₂O were mixed to obtain total volume of 20 μl. A mastermix was created with 4.2 μl RNAse-Free H₂O, 2 μl 10×RT Buffer, 0.8 μl25×dNTPs, and 2 μl Random Primers for each reaction (optionally, 10%more can be added to the total number of reactions to be performed toensure sufficient volume). Reverse Transcriptase (1 μl) was added foreach reaction and mixed carefully without vortexing. Some duplicatesamples (˜10% of total) were assigned to an RT(−) set and enough mastermix lacking Reverse Transcriptase was prepared to include thesecontrols. Master mix (20 μl) was added (or RT(−) master mix) to 20 μl offixed-input RNA sample. The reactions were incubated at 37° C. for 2hours, heated to 85° C. for 5 min., and placed on ice. Samples of cDNAwere stored at 4° C. if used by the next day or stored at −20° C. forlonger periods.

RT-PCR.

For PCR analysis, the kit utilized was TaqMan® Fast Universal Master Mix(2×), No AmpErase® UNG. For reactions to be run on ABI 7500, 2 μl ofcDNA template or RT(−) control template was placed in each well. Amaster mix was made including 1.0 μl TaqMan Expression Assay(primers/probes) and 7 μl Nuclease-Free H₂O for each sample to be run,and 18 μl of master mix+Expression Assay+Nuclease-Free H₂O was added toeach 2 μl sample, mix by pipetting. The plate was sealed, spun down andloaded in an ABI7500. At least one housekeeping mRNA Expression Assay(e.g. GAPDH; Mm03302249_g1 or Hs02758991_g1) was included in each set ofRNA samples to be assessed.

The effect of Cry modulator Compound 72 on core clock function wasexamined in diabetic mice and non-diabetic mice. Male C57Bl/6J DIO miceat 17 weeks of age (The Jackson Laboratory, Sacramento, Calif.) weremaintained on a high fat diet (HFD) and acclimated for a 2 week periodbefore treatment to recapitulate diabetes and obesity. Three mice weretreated with compound or vehicle for each of the 5 time points. Micewere dosed with vehicle (water for injection (WFI) or the Compound 72(50 mg/kg in WFI) at a dose volume of 5 ml/kg, BID (twice daily), viaoral gavage. In total, each mouse received 5 doses of compound over thecourse of the experiment. Mice were weighed and randomly assigned toeach treatment based on weight the evening before the commencement ofthe study. Beginning on Day 3 at 3:00 PM, a group of animals from thevehicle and Compound 72-treated groups were euthanized using CO₂asphyxiation, 50 mg of liver, epididymal white fat, and skeletal muscle,were excised for RNA preparation.

The effect of Compound 72 was also examined in normal Balb/c mice oncircadian mRNAs over 24 hours. The mice were 8 weeks old, obtained fromCharles River, and allowed to acclimate for 2 weeks. Mice were dosed BIDfor 3 days, with a total of 7 doses for each animal, with the last dose12 hours before obtaining tissues. Mice were dosed with vehicle (WFI) orCompound 72 (50 mg/kg in WFI) at a dose volume of 5 ml/kg, BID, via oralgavage. Beginning on Day 3 at 3:00 PM, a group of animals from thevehicle and Compound 72 groups were sacrificed, and approximately 50 mgof liver, lung, kidney, adrenal gland, spleen, epididymal fat, and brownadipose tissue were excised and placed in RNALater.

The core clock mRNAs from vehicle-treated C57Bl/6J DIO mice displayedthe characteristic circadian expression pattern (FIG. 1). With Compound72 treatment, however, Per2 mRNA was suppressed in both C57Bl/6J DIO andBalb/c mice over 24 hours, and these were most reduced at ZT8 and againat ZT8 24 hours later (FIGS. 1 A & B). Bmal1 mRNAs were substantiallyincreased by Compound 72 treatment at ZT8 and 32 hours after theoriginal ZT0 in both strains of mice. Bmal1 transcripts also displayed aprominent phase delay in C57Bl/6J DIO mice and to a lesser extent inBalb/c mice (FIGS. 1C & D). The mRNA for Cry1 was suppressed during itspeak of expression during the dark period (FIGS. 1E & F, shadedregions). The phase advance in vehicle-treated Cry1 transcripts observedin DIO mice relative to vehicle-treated Balb/c mice may be in part dueto the known effects of high fat diet on many diurnal patterns(Eckel-Mahan et al. (2013) Cell). In contrast with Cry1 mRNA, the mRNAfor Cry2 peaked in the daytime hours in vehicle-treated mice, but thiswas strongly attenuated by treatment with Compound 72 in both C57Bl/6JDIO and Balb/c mice (FIGS. 1G & H).

In the same 24 hour studies described above, the circadian pattern ofmRNAs for the gluconeogenic genes Pck1 (PEPCK) and G6Pc (Glucose6-phosphatase catalytic subunit) were substantially altered by Compound72 relative to vehicle in C57Bl/6J DIO mice (FIGS. 2A & C). Thevehicle-treated mice displayed a pattern that was flattened andphase-advanced over that observed for chow-fed C57Bl/6J mice in otherstudies (Hughes et al. (2009)). The Compound 72-treated C57Bl/6J DIOmice displayed a peak of expression for both of these gluconeogenicgenes in the early evening (ZT14), which is closer to the peak time oftheir expression observed in chow-fed mice (Hughes et al. (2009)). Thediurnal patterns of expression for Pck1 and G6Pc were altered more byCompound 72 in the C57Bl/6J DIO mice relative to vehicle than they werein the Balb/c mice (FIGS. 2B & D).

The effects of multiple Cry modulator compounds, Compound 72, Compound48 Compound 9 and Compound 57 were examined in ICR mice. The mice weretreated with 50 mg/kg of each compound (Compound 72, Compound 48,Compound 9, and Compound 57), PO (oral administration) at a dose volumeof 5 ml/kg for 4 days, BID or vehicle control. Each compound caused asuppression of liver Per2 expression (FIG. 3A). Compound 48 treatmentresulted in an 8-fold increase in Bmal1 mRNA at ZT6, Compound 72 andCompound 9 treated mice displayed a 4 fold increase, and Compound 57treated mice displayed at least a 2 fold increase (FIG. 3B). Compound72, Compound 48 and Compound 9 also caused a decrease in Cry2transcripts (FIG. 3C).

The levels of core clock gene mRNAs in whole blood may provide anon-invasive method of determining effects of compounds in treatedsubjects. Male db/db mice (9 weeks of age, The Jackson Laboratory, BarHarbor, Me.) were used with n=8 for each experimental group. Mice weredosed with Compound 72 (100 mg/kg, P.O; dose volume 5 ml/kg, in 10%Kolliphor), or 10% Kolliphor, once daily for three days at ZT0 (7:00 am)(ZT refers to Zeitgeber Time, and indicates the time at which the lightswere turned on to stimulate day in the mouse facility). On the final dayat ZT7.5 (2:30 pm), animals were euthanized using CO₂ asphyxiation, andthe blood collected from the heart via cardiac puncture. Whole blood wastransferred to a tube containing RNALater for assay by RT-qPCR.

The D-box binding protein Dbp is regulated in a strongly circadianmanner. Compound 72 caused a statistically significant suppression ofDbp gene expression in this study (FIG. 4), demonstrating that whiteblood cells in mice treated with such compounds can provide informationabout the effects of compounds on the core clock mechanism in the wholeorganism. Such information may be used as a diagnostic marker, or as abiomarker to assess the effects of Cry modulators and other therapeuticagents that impact the core circadian mechanism.

For compounds that interact directly with the core clock mechanism, thetime of dosing may be critical for maximizing their effects. Db/db micewere treated with a single dose of Compound 72 (50 mg/kg) given ateither ZT0 or ZT10. The former (ZT0) is coincident with the peak of Cry1and Bmal1 protein in mouse liver and the latter (ZT10) corresponds tothe approximate nadir of Cry1 and Bmal1. Liver tissue was taken 7.5hours later for each, and the samples were examined for core clock genemRNAs by RT-qPCR. Since Compound 72 had a greater relative impact on theclock mRNAs at the peak compared to the nadir (FIG. 5), dosing aroundthe time of the former provides for greater effects on the clockmechanism, and this may further lead to a greater effect on themetabolic outputs of the clock.

Example 6 Effect of Single Dose of Compound 72 Administered at Peak orNadir of Clock Gene Expression in a Diabetes Mouse Model

The effect of Compound 72 on glucose metabolism was assessed whenadministered as a single dose administered either at peak or nadir ofcore clock genes Cry1/Bmal1 expression in a db/db mouse model of type IIdiabetes.

Male db/db mice homozygous for Lepr^(db) (6 weeks of age), were obtainedfrom The Jackson Laboratory (Bar Harbor, Me.). Mice were group housed ona normal light/dark cycle (lights on: 07:00-19:00 h) with ad libitumaccess to a standard pelleted mouse diet and water. Animals wereaccustomed to these conditions for 2 weeks before experimentation. Micewere split into two study arms, to be dosed at either the peak or nadirof Cry1 and Bmal1 gene expression. Mice were dosed once with vehicle(10% Kolliphor, Sigma-Aldrich) or Compound 72 (50 mg/kg in 10% Kolliphorin water) at a dose volume of 5 ml/kg, QD, via oral gavage, at ZT0(peak, 7:00 am) or ZT10 (nadir, 5:00 pm). Animals were weighed on day 0and randomly assigned to treatment groups so that each group had similaraverage starting weights. Mice dosed at ZT0 were fasted overnightbeginning at 10:30 pm, when they were transferred to a clean cage andgiven free access to water, but not food, for a period of 12 hours. Micedosed at ZT10 were fasted beginning at 8:30 am in the same manner. Onthe day of the study, after dosing, mice underwent a tail cut injury 2hours prior to measurement of fasting blood glucose, to allow recoveryfrom any stress the procedure might cause. Fasting blood glucose (FBG)was assessed from the animals at 10:30 am or 8:30 pm using an AlphaTRAKglucometer (Abbott Laboratories, USA). At 11:30 am (peak dosed mice) or9:30 pm (nadir dosed mice) each animal was dosed with 0.5 g/kg ofglucose, then blood glucose was measured at t=15, 30, 60, 90 and 120minutes after glucose load. Animals were terminated following the lastblood collection and tissues and blood harvested for other endpointdeterminations.

Fasting blood glucose values and glucose measurements taken during theOGTT were averaged and graphed (GraphPad Prism, GraphPad Software, LaJolla, Calif.). The area under the curve (AUC) was calculated for eachindividual animal. Statistical analysis was performed using one-wayANOVA followed by the appropriate post-test. Significance was acceptedwhen p<0.05. Data are presented as mean and S.E.M.

Administration of Compound 72 (50 mg/kg, PO) at a single dose,administered at the peak of Cry1 and Bmal1 gene expression, to db/dbmice resulted in an apparent effect on the OGTT measurement (FIG. 6A)compared to the vehicle control, but no effect when dosed at the nadir(FIG. 6B). The AUC calculated from the OGTT of animals dosed at the peakof gene expression showed that Compound 72 treatment resulted in areduction in glucose excursion of 14% (74098+/−4194 to 63842+/−4318;FIG. 7).

Example 7 Effect of a Single Dose of Compound 72 Administered Over 7Days in a Diabetes Mouse Model

The effect of Compound 72 on glucose metabolism and insulin levels wasassessed when administered as a single dose administered over 7 days ina db/db mouse model of type II diabetes.

Male db/db mice homozygous for Lepr^(db) (6 weeks of age), were obtainedfrom The Jackson Laboratory (Bar Harbor, Me.). Mice were group housed ona normal light/dark cycle (lights on: 07:00-19:00 h) with ad libitumaccess to a standard pelleted mouse diet and water. Animals wereaccustomed to these conditions for 2 weeks before experimentation. Micewere dosed with vehicle (10% Kolliphor, Sigma-Aldrich) or Compound 72(50 mg/kg in 10% Kolliphor in water) at a dose volume of 5 ml/kg, QD,via oral gavage, at ZT0 (7:00 am) for seven days. Mice were weighed onDay 0 and randomly assigned to either treatment group so that each grouphad similar average starting weights. At 10:30 pm on the evening priorto endpoint measurement, mice were placed into clean cages and givenfree access to water, but not food for a period of 12 hours before thefasting blood glucose measurement. On the final day of the study,animals were dosed as normal, and then underwent a tail cut injury 2hours prior to measurement of fasting blood glucose, to allow recoveryfrom any stress the procedure might cause. Fasting blood glucose (FBG)was assessed from the animals at 10:30 am using an AlphaTRAK glucometer(Abbott Laboratories, USA). Following the FBG measurement blood wascollected from each mouse, using a tail milking technique, into acapillary tube. Capillary tubes were centrifuged in a hematocrit (BDTriac 0200) and the resultant plasma transferred to an eppendorff. Thissample, labelled as t=0 hr, was frozen at −80° C. to allow latermeasurement of insulin. At 11:30 am each animal was dosed with 0.5 g/kgof glucose, then blood glucose was measured at t=15, 30, 60, 90 and 120minutes after glucose load. At the end of the OGTT blood was collectedfor a t=2 hr insulin determination as described above. Animals wereterminated following the last blood collection and tissues and bloodharvested for other endpoint determinations (detailed elsewhere). Plasmaand liver tissue from Compound 72 treated animals were submitted to aCRO for measurement of compound levels, using LC/MS/MS and comparing toa standard curve of known compound amounts.

Fasting blood glucose values and glucose measurements taken during theOGTT were averaged and graphed (GraphPad Prism, GraphPad Software, LaJolla, Calif.). The area under the curve (AUC) was calculated for eachindividual animal. Plasma insulin levels were determined using anUltrasensitive Insulin ELISA (ALPCO, Salem, N.H.). The HOMA-IR(homeostatic model assessment-insulin resistance) was calculated usingthe following formula: (FPI (μU/L)×FPG (mmol/L))/22.5, where FPI and FPGdenote Fasting Plasma Insulin and Fasting Plasma Glucose, respectively.Insulin data was also represented in GraphPad Prism format. Statisticalanalysis was performed using one-way ANOVA followed by the appropriatepost-test. Significance was accepted when p<0.05. Data are presented asmean and S.E.M.

Administration of Compound 72 (50 mg/kg, PO) for 7 days to db/db miceresulted in a significant reduction in FBG compared to vehicle control(484.9+/−34.37 mg/dL to 385.0+/−29.69 mg/dL; FIG. 8A). During the courseof the OGTT measurement, Compound 72 treated animals were lower than thevehicle control group (FIG. 8B). The AUC calculated from the OGTT showedthat Compound 72 administration resulted in a significant reduction inglucose excursion (54845+/−4112 to 35942+/−3192; FIG. 8C).

Plasma insulin measurements were made from the samples taken at t=0 andt=2 hr, and are shown in FIG. 9A. Insulin was reduced by treatment withCompound 72 at both t=0 and t=2 hr time-points by 20% and 21%,respectively (4.70+/−0.76 to 3.78+/−0.69 ng/mL and 3.17+/−0.67 to2.53+/−0.50 ng/mL, respectively). The HOMA-IR (an indication ofre-sensitization to insulin) was reduced by 33% from 139.91+/−26.57 to93.69+/−23.60 units (FIG. 9B).

Compound 72 compound levels were assessed from the samples taken atstudy termination, and are shown in FIG. 10, along with the EC₅₀ valuedetermined from the Per2 assay as described in Example 3, for comparisonpurposes. Compound 72 was found in both the plasma and liver at around 8hours after administration of the last dose (plasma: 0.53+/−0.03 μM;liver: 0.67+/−0.05 μM, shown as Mean and S.E.M). In both cases compoundslevels were slightly higher than the Per2 EC₅₀ value determined forCompound 72 (0.4 μM; 1.3 fold and 1.7 fold higher than the EC₅₀ value inplasma and liver, respectively).

Example 8 Effect of Increasing Dosages of Compound 72 in a DiabetesMouse Model

The effect of Compound 72 was assessed over increasing dosesadministered over 7 days on glucose metabolism and insulin levels in adb/db mouse model of type II diabetes.

Male db/db mice homozygous for Lepr^(db) (5 weeks of age), were obtainedfrom The Jackson Laboratory (Bar Harbor, Me.). Mice were group housed ona normal light/dark cycle (lights on: 07:00-19:00 h) with ad libitumaccess to a standard pelleted mouse diet and water. Animals wereaccustomed to these conditions for 2 weeks before experimentation. Micewere dosed with vehicle (10% Kolliphor, Sigma-Aldrich) or Compound 72 at10, 50 or 100 mg/kg (in 10% Kolliphor in water) at a dose volume of 5ml/kg, QD, via oral gavage, at ZT0 (7:00 am) for seven days. Theexperimental methods performed were the same as those detailed inExample 11. The compound levels in plasma and liver tissue from Compound72 treated animals were measured using LC/MS/MS and compared to astandard curve of known compound amounts.

Administration of Compound 72, at ascending doses, for 7 days to db/dbmice resulted in a reduction in fasting blood glucose levels compared tothe vehicle control group at 50 and 100 mg/kg, though this did not reachstatistical significance (vehicle control: 491.0+/−51.30 mg/dL, 50mg/kg: 402.8+/−25.25 mg/dL, 100 mg/kg: 420.7+/−26.44 mg/dL; FIG. 11A).Compound 72, administered at a dose of 10 mg/kg, demonstrated noreduction in fasting blood glucose levels (503.5+/−49.68 mg/dL). Duringthe course of the OGTT measurement, Compound 72 treated animalsdemonstrated a dose dependent effect on glucose excursion from theanimals following a glucose load (FIG. 11B). The area under the curvecalculated from the OGTT showed that Compound 72 administration reducedthe glucose AUC in a dose dependent manner which was significant at 100mg/kg (vehicle: 46088+/−3303, 10 mg/kg: 39771+/−4244, 50 mg/kg:35527+/−3215, 100 mg/kg: 28499+/−3079; FIG. 11C).

Plasma insulin measurements were made from the samples taken at t=0 andt=2 hr, and are shown in FIG. 12A. Insulin was reduced, at t=0, bytreatment with Compound 72 when dosed at 100 mg/kg (from 5.68+/−0.43 to4.63+/−0.17 ng/mL (vehicle and 100 mg/kg group, respectively)). TheHOMA-IR was reduced in a dose dependent manner following Compound 72administration (vehicle: 169.3+/−18.41, 10 mg/kg: 172.7+/−16.61, 50mg/kg: 149.2+/−16.49, 100 mg/kg: 111.9+/−9.02 units; FIG. 12B). At 100mg/kg, the effect of Compound 72 was significant (34% reduction comparedto the vehicle control group).

Compound 72 compound levels were assessed from the samples taken atstudy termination, and are shown in FIG. 13, along with the Per2 EC₅₀value (as described in Example 3), for comparison purposes. Compound 72was found in both the plasma and liver at around 8 hours afteradministration of the last dose, with exposure levels which wereincreased relative to the increase in dose administered (plasma, 10mg/kg: 0.09+/−0.01 μM; 50 mg/kg: 0.57+/−0.03 μM; 100 mg/kg: 1.22+/−0.17μM; liver, 10 mg/kg: 0.12+/−0.01 μM; 50 mg/kg: 0.78+/−0.06 μM; 100mg/kg: 1.81+/−0.22 μM). In both plasma and liver exposure levels were1.4 fold and 1.95 fold higher at 50 mg/kg, and 3 fold and 4.5 foldhigher at 100 mg/kg, than the Per2 EC₅₀ value in the plasma and liverrespectively.

Example 9 Effect of Increasing Dosages of Compound 9 in a Diabetes MouseModel

The effect of Compound 9 was assessed over increasing doses administeredover 7 days on glucose metabolism and insulin levels in a db/db mousemodel of type II diabetes.

Male db/db mice homozygous for Lepr^(db) (5 weeks of age), were obtainedfrom The Jackson Laboratory (Bar Harbor, Me.). Mice were group housed ona normal light/dark cycle (lights on: 07:00-19:00 h) with ad libitumaccess to a standard pelleted mouse diet and water. Animals wereaccustomed to these conditions for 2 weeks before experimentation. Micewere dosed with vehicle (10% Kolliphor, Sigma-Aldrich) or compoundCompound 9 at 30, 100 or 300 mg/kg, or Rosiglitazone at 30 mg/kg (in 10%Kolliphor in water) at a dose volume of 5 ml/kg, QD, via oral gavage, atZT0 (7:00 am) for seven days. Rosiglitazone is an anti-diabetictherapeutic agent that was used for positive control. The experimentalmethods performed were the same as those detailed in Example 11. Thecompound levels in plasma and liver tissue from Compound 9 treatedanimals were measured using LC/MS/MS and compared to a standard curve ofknown compound amounts.

Administration of Compound 9, at ascending doses, for 7 days to db/dbmice resulted in a reduction in fasting blood glucose levels at 100mg/kg compared to the vehicle control group (from 492.8+/−48.07 to403.1+/−39.73 mg/dL; FIG. 14A) but no statistically significant effectoverall. Compound 9, administered at a dose of 30 and 100 mg/kg,demonstrated a dose dependent reduction in glucose levels measurementduring the OGTT, with no increased effect observed at the highest dosetested of 300 mg/kg (FIG. 14B). The area under the curve calculated fromthe OGTT showed that administration of Compound 9 reduced the glucoseAUC in a dose dependent manner which was significant at both 100 and 300mg/kg (vehicle: 56046+/−3204, 30 mg/kg: 44442+/−3895, 100 mg/kg:33643+/−4822, 300 mg/kg: 33650+/−4688; FIG. 8C). The glucose AUC wasreduced by 21%, 40% and 40% at 30, 100 and 300 mg/kg, respectively, fromthe vehicle control group. Rosiglitazone, used as a positive control forthe animal model, significantly inhibited fasting blood glucose (from492.8+/−48.07 to 280.4+/−13.66 mg/dL; FIG. 14A), and glucose AUC (from56046+/−3204 to 11502+/−2118 units; FIG. 14C).

Plasma insulin measurements were made from the samples taken at t=0 andt=2 hr, and are shown in FIG. 15A. Insulin was reduced followingtreatment with Compound 9 at both t=0 (vehicle: 14.89+/−2.93, 30 mg/kg:10.94+/−1.62, 100 mg/kg: 7.71+/−1.26, 300 mg/kg: 10.54+/−1.6 ng/mL) andt=2 hr (vehicle: 7.44+/−0.92, 30 mg/kg: 5.76+/−0.11, 100 mg/kg:3.70+/−0.29, 300 mg/kg: 4.01+/−0.44 ng/mL). The HOMA-IR was reduced in adose dependent manner following administration of Compound 9, thoughthere was a lesser effect of the compound at 300 mg/kg on this endpoint(vehicle: 438.8+/−87.88, 30 mg/kg: 289.9+/−24.40, 100 mg/kg:175.3+/−27.52, 300 mg/kg: 301.4+/−52.66 units; FIG. 15B). At 100 mg/kg,the effect of Compound 9 was significant (60% reduction compared to thevehicle control group). Rosiglitazone reduced insulin levels at t=0 andt=2 hr (to 3.05+/−0.14 and 2.28+/−0.08 ng/mL, respectively; FIG. 15A),as well as significantly reducing the HOMA-IR (to 50.58+/−3.52 units,FIG. 15B).

Compound 9 tissue levels were assessed from the liver samples taken atstudy termination, and are shown in FIG. 16, along with the Per2 EC₅₀value, for comparison purposes. Compound 9 was found in the plasma liverat around 8 hours after administration of the last dose, with exposurelevels which were increased relative to the increase in doseadministered between the 30 mg/kg and 100 mg/kg doses. Exposure levelsat 300 mg/kg indicated an accumulation of drug (7.1 fold increase ratherthan 3 fold as expected). Compound levels in the liver samples ofanimals administered 30, 100 or 300 mg/kg of Compound 9 were 0.19+/−0.02μM, 0.67+/−0.05 μM and 4.77+/−1.06 μM, respectively. Liver exposurelevels following administration of 30 mg/kg Compound 9 were around 1.6fold below the Per2 EC₅₀ value (0.3 μM), while levels were 2.2 fold and15.9 fold higher at 100 mg/kg and 300 mg/kg, respectively.

Example 10 Effect of Compound 72 in a Diet-Induced Obesity Mouse Model

The effect of Compound 72 was examined in a diet-induced obesity (DIO)mouse model of type II diabetes.

Male C57/Bl6J DIO mice were obtained from The Jackson Laboratory(Sacramento, Calif.). Mice were group housed on a normal light/darkcycle (lights on: 07:00-19:00 h) with ad libitum access to high fat diet(D12492 (60 kcal % fat), Research Diets, Inc.) and water. Animals wereaccustomed to these conditions for at least 2 weeks beforeexperimentation and used at approximately 24 weeks of age. Mice weredosed with vehicle (10% Kolliphor, Sigma-Aldrich), Compound 72 (100mg/kg in 10% Kolliphor in water) or Rosiglitazone (30 mg/kg in 10%Kolliphor in water) at a dose volume of 5 ml/kg, QD, via oral gavage, atZT0 (7:00 am) for seven days. Rosiglitazone is an anti-diabetictherapeutic agent that was used for positive control. Mice were weighedon Day 0 and randomly assigned to either treatment group so that eachgroup had similar average starting weights. At 10:30 pm on the eveningprior to endpoint measurement, mice were placed into clean cages andgiven free access to water, but not food for a period of 12 hours beforethe fasting blood glucose measurement. On the final day of the study,animals were dosed as normal, and then underwent a tail cut injury 2hours prior to measurement of fasting blood glucose, to allow recoveryfrom any stress the procedure might cause. Fasting blood glucose (FBG)was assessed from the animals at 10:30 am using an AlphaTRAK glucometer(Abbott Laboratories, USA). Following the FBG measurement blood wascollected from each mouse, using a tail milking technique, into acapillary tube. Capillary tubes were centrifuged in a hematocrit (BDTriac 0200) and the resultant plasma transferred to an eppendorff. Thissample, labelled as t=0 hr, was frozen at −80 to allow later measurementof insulin. At 11:30 am each animal was dosed with 1.5 g/kg of glucose,then blood glucose was measured at t=15, 30, 60, 90 and 120 minutesafter glucose load. At the end of the OGTT blood was collected for a t=2hr insulin determination as described above. Animals were terminatedfollowing the last blood collection and tissues and blood harvested forother endpoint determinations.

Fasting blood glucose values and glucose measurements taken during theOGTT were averaged and graphed (GraphPad Prism, GraphPad Software, LAJolla, Calif.). The area under the curve (AUC) was calculated for eachindividual animal. Plasma insulin levels were determined using anUltrasensitive Insulin ELISA (ALPCO, Salem, N.H.). The HOMA-IR(homeostatic model assessment-insulin resistance) was calculated usingthe following formula: (FPI (μU/L)×FPG (mmol/L))/22.5, where FPI and FPGdenote Fasting Plasma Insulin and Fasting Plasma Glucose, respectively.Insulin data was also represented in GraphPad Prism format. Statisticalanalysis was performed using one-way ANOVA followed by the appropriatepost-test. Significance was accepted when p<0.05. Data are presented asmean and S.E.M.

Administration of Compound 72 (100 mg/kg, PO) for 7 days to C57/Bl6J DIOmice resulted in a significant reduction in fasting blood glucose levelscompared to vehicle control (237.2+/−15.29 mg/dL to 177.1+/−8.28 mg/dL;FIG. 17A). During the course of the OGTT measurement, Compound 72treated animals were much lower than the vehicle control group (FIG.17B). The AUC calculated from the OGTT showed that Compound 72administration resulted in a significant reduction in glucose excursion(31511+/−1670 to 17055+/−769.1; FIG. 17C). Rosiglitazone, used as apositive control for the animal model, reduced fasting blood glucose to153.9+/−5.05 mg/dL and the glucose AUC to 11500+/−1104 units.

Plasma insulin measurements were made from the samples taken at t=0 andt=2 hr, and are shown in FIG. 18A. Insulin was reduced followingtreatment with Compound 72 at both t=0 (vehicle: 5.00+/−0.92, 100 mg/kg:3.12+/−0.24, ng/mL) and t=2 hr (vehicle: 4.82+/−0.60, 100 mg/kg:2.88+/−0.21 ng/mL). The HOMA-IR was significantly reduced followingCompound 72 administration (vehicle: 70.76+/−11.30, 100 mg/kg:32.54+/−3.37 units; FIG. 18B). Rosiglitazone (30 mg/kg) reduced insulinat t=0 and t=2 hr (to 2.70+/−0.12 and 2.30+/−0.06 ng/mL, respectively)and significantly reduced HOMA-IR (to 24.60+/−1.42 units).

Example 11 Effect of Compound 72 on the Development of Cortisone InducedInsulin Resistance in Rats

Repeated administration of cortisone to rats for 6 days induces asignificant decrease in body weight which is associated with asignificant rise in plasma insulin and glucose. These effects aremediated via cortisol generated by 11-βHSD1 activity. Glucocorticoidreceptor antagonists such as mifepristone ameliorate the effects ofcortisol on insulin resistance. The aim of these experiments was todetermine the effect of Compound 72 on the development ofcortisone-induced insulin resistance in the rat.

Animals were dosed with cortisone (30 mg/kg sc qd) in combination withtest compounds for 6 days and then terminated 27 hours after the finaldose of cortisone. A reference standard (Mifepristone) was alsoincluded. Cortisone 21-acetate (Sigma C-3130) was supplied by RenaSciand administered using a dose volume of 5 ml/kg via the subcutaneousroute as a fine suspension in 1% methylcellulose. Compound 72 (50 mg/kgin 10% Kolliphor in water) was dosed QD using a dose volume of 5 ml/kgvia oral gavage. Mifepristone (Sigma M8046) was provided by RenaSci.

Glucose and insulin determinations were performed on plasma samplesobtained from a tail vein bleed taken after a 12 h fast, approximately27 h after the last cortisone dose. Animals were then terminated and aterminal (cardiac) blood sample taken from which plasma was prepared.

Thirty four male Sprague Dawley rats (weight range 200-250 g) wereordered from Charles River, Margate, Kent, UK. Rats were group housed ona normal light/dark cycle (lights on: 07:00-19:00 h) with free access toa standard pelleted rat diet and tap water at all times. Animals wereaccustomed to these conditions for 2 weeks before experimentation.Subsequently, animals underwent a 3 day baseline period during whichthey were dosed once daily with vehicle at t=0 h (07:00). This procedurehas been found to reduce the incidence of stress-related effects instudies. All drugs were administered for 6 days as shown in Table 2below. Body weight was recorded immediately before dosing began at 07:00(t=0 h). Cortisone was administered via the subcutaneous route (sc)whilst Compound 72 and mifepristone were administered orally, viagavage, immediately after cortisone administration at t=0 h.

TABLE 2 Group Treatment(t = 0 h; 07:00) n A Vehicle (1% methylcellulose;Vehicle (5 ml/kg po) 8 5 ml/kg sc) B Cortisone (30 mg/kg sc) Vehicle (5ml/kg po) 8 C Cortisone (30 mg/kg sc) Compound72 (50 mg/kg po) 8 DCortisone (30 mg/kg sc) Mifepristone (30 mg/kg po) 8

On Day 6 of dosing rats were fasted for 12 h beginning at 22:30 (timedto coincide with the Day 7 termination). On Day 7 rats were administeredvehicle but not cortisone (sc) followed by oral administration ofvehicle/Compound 72/mifepristone as usual at 07:00. At 10:30 on Day 7,27 h after the final dose of cortisone, a blood sample (300 μl) wastaken from the lateral tail vein into tubes containing EDTA (Sarstedt16.444). The blood was centrifuged and resultant plasma aliquot storedat −75° C. The animals were euthanized by CO₂ asphyxiation followed bycervical dislocation. Terminal blood (approximately 10 ml) was collectedby cardiac puncture into tubes containing EDTA (Sarstedt 5 ml 32.332)and then centrifuged and plasma stored at −75° C. The tail vein plasmawas analyzed for glucose (n=2) using a commercial clinical reagent(Thermoelectron Infinity glucose reagent (TR15421) and for insulin (n=1)using the Mercodia ultrasensitive rat insulin rat ELISA (10-1251-10).

Plasma glucose and insulin were analyzed by robust regression or generallinear model with treatment as a factor and bleeding order and baselinebody weight as covariates. A log transformation was used if appropriate.Appropriate multiple comparison tests (two-tailed) were used todetermine significant differences from the vehicle group and from theCortisone group. P<0.05 was considered to be statistically significant.

Administration of Compound 72 to rats significantly reduced the increasein plasma glucose and insulin caused by cortisone administrationtreatment. Plasma glucose levels were increased from 6.28+/−0.30 mM to10.17+/−0.51 mM following treatment with cortisone, which wassignificantly reduced by Compound 72 (50 mg/kg) to 8.55+/−0.3 mM(p<0.01; Mean and S.E.M). Plasma insulin levels were increased from0.70+/−0.11 ng/mL to 8.19+/−0.91 ng/mL upon cortisone treatment, whichwas reduced by Compound 72 (50 mg/kg) to 5.24+/−1.11 ng/mL (p<0.05; datarepresented as Mean and S.E.M; FIG. 19). Mifepristone, used as apositive control for the animal model, significantly reduced plasmaglucose and plasma insulin to 7.43+/−0.27 ng/mL and 3.62+/−0.29 ng/mL,respectively.

HOMA-IR values were calculated as described in Compound 9, and the datais shown in FIG. 20. Cortisone treatment increased HOMA-IR to95.57+/−11.4 units compared with the vehicle: vehicle control group(5.27+/−1.04 units). Administration of Compound 72 (50 mg/kg) andmifepristone significantly reduced the HOMA-IR value in insulinresistant rats to 56.94+/−11.18 units and 29.99+/−2.54 units,respectively.

Example 12 Pharmacokinetic (PK) Analysis of Compound 9 and Compound 72

Male ICR mice (weighing 30-40 g, Charles River Laboratories) were usedfor the experiment with n=3 mice for each experimental group (27 micetotal for the study). Mice were dosed with Cry Modulator Compound 9 orCompound 72 (50 mg/kg, P.O; dose volume 5 ml/kg, in 10% Kolliphor).Blood and liver tissue were collected at the following time points afteradministration: 15, 30, 60, 90 minutes, 3, 6, 12 and 24 hours. A controlgroup of animals (TO) was also sampled. Animals were euthanized with CO₂and the blood collected from the heart using cardiac puncture,transferred to an EDTA tube, and then centrifuged at 5400 rpm for 5minutes at 4° C. The resultant plasma was frozen using dry ice and thenstored at −80° C. until ready for assay. Liver tissue was removed fromeach animal, 0.5 g was collected in an eppendorff, frozen and submittedfor pharmacokinetic measurement. Plasma and liver tissue from eachanimal was submitted to a CRO for measurement of compound levels, usingLC/MS/MS and comparing to a standard curve of known compound amounts inboth plasma and liver. Raw data was analyzed using WinNonLin for PKparameters (Cmax, Tmax, elimination t^(1/2), MRT (mean residence time),AUC (area under the curve)−(0−last and % extrapolated).

Male SD rats (weighing 250-300 g, Charles River Laboratories) were usedfor the experiment with n=4 rats for each experimental group. Rats weredosed with Compound 72 (50 mg/kg, P.O; dose volume 5 ml/kg, in 10%Kolliphor). Blood was collected at the following time points afteradministration: 15, 30, 60, 90 minutes, 3, 6, 12 and 24 hours. Apre-dose sample was also collected. Animals were cannulated by technicalstaff at Charles River, prior to delivery at Reset. Whole blood (0.3 ml)was collected from a cannula in the right common jugular vein at eachtime-point. The whole blood was transferred to an EDTA tube, and thencentrifuged at 5400 rpm for 5 minutes at 4° C. The resultant plasma wasfrozen using dry ice and then stored at −80° C. until ready for assay.0.9% Sodium Chloride (0.3 ml) was administered for fluid replenishmentafter each blood draw. 0.1 ml of Sodium Heparin (500 IU/ml) was used asa lock solution after the 12 hr time-point. Samples were assayed asdescribed above. Tables 3 and 4 summarize the results from the analyses.

TABLE 3 PK Parameters of Compound 9 and Compound 72 PK properties MRTMRT Cmax Tmax Elimination (0-last, (0-last; AUC (total; % AUC (ng/ml)(hr) T1/2 (hr) hr) ng.hr/mL) ng.hr/mL) extrapolated Compound Compound 9Plasma Mouse 893 0.25 3.18 3.92 1920 1930 0.564 Compound 9 Liver Mouse33000 0.25 3.12 1.50 26800 26800 0.142 Compound 72* Plasma Mouse 11490.25 3.13 5.30 4158 4236 1.74 +/− − +/− +/− +/− +/− +/− 108.7 0.5 0.240.39 249.7 264.1 1.20 Compound 72* Liver Mouse 7890 0.25 3.40 4.74 1782217961 0.85 +/− − +/− +/− +/− +/− +/− 972.6 0.5 0.28 0.51 2187 2184 0.24Compound 72 Plasma Rat 7098 1.7 1.87 4.94 57933 57952 3.3 *Denotes datafrom 4 experiments, shown as Mean and S.E.M.

TABLE 4 Unbound Exposure Data Cmax Cmax Total Unbound Total Unbound CmaxUB Cmax UB Plasma Plasma Liver Liver Compound (plasma) (plasma) (liver)(liver) AUC AUC AUC AUC (ng/ml) (ng/ml) (ng/ml) (ng/ml) AUC (0- AUC (0-AUC (0- AUC (0- last) last) last) last) Compound 9 893 26.8 33000 9901910 57 26800 804 Compound 72 1149 41.93 7890 288.0 4158 151.5 17822650.5 (m)* +/− +/− +/− +/− +/− +/− +/− +/− 108.7 3.97 972.6 35.51 249.79.11 2187 79.81 Compound 72 7098 277 57933 2259 (r) *denotes data from 4experiments, shown as Mean and S.E M.

Example 13 Effect of Increasing Dosages of Compound 72 in a Diet-InducedObesity Mouse Model

The effect of Compound 72 was assessed over increasing doses in adiet-induced obesity (DIO) mouse model of type II diabetes.

Male C57/Bl6J DIO mice were obtained from The Jackson Laboratory(Sacramento, Calif.). Mice were group housed on a normal light/darkcycle (lights on: 07:00-19:00 h) with ad libitum access to high fat diet(D12492 (60 kcal % fat), Research Diets, Inc.) and water. Animals wereaccustomed to these conditions for at least 2 weeks beforeexperimentation and used at approximately 26 weeks of age. Mice weredosed with vehicle (10% Kolliphor, Sigma-Aldrich), Compound 72 (10, 30or 100 mg/kg in 10% Kolliphor in water) or Rosiglitazone (30 mg/kg in10% Kolliphor in water) at a dose volume of 5 ml/kg, QD, via oralgavage, at ZT0 (7:00 am) for seven days. The experimental methodsperformed were the same as those detailed in Example 10.

Administration of Compound 72, at ascending doses, for 7 days toC57/Bl6J DIO mice resulted in a reduction in fasting blood glucoselevels compared to vehicle control which reached significance at 100mg/kg (vehicle control: 226.9+/−13.11 mg/dL, 10 mg/kg: 206.8+/−8.36mg/dL, 30 mg/kg: 197.5+/−12.06 mg/dL, 100 mg/kg: 176.3+/−7.83 mg/dL,FIG. 22A. During the course of the OGTT measurement, Compound 72 treatedanimals demonstrated a reduction in glucose excursion following aglucose load (FIG. 22B. The area under the curve calculated from theOGTT showed that Compound 72 administration reduced the glucose AUC,demonstrating significance at 30 and 100 mg/kg (vehicle: 26090+/−1917,10 mg/kg: 22563+/−1224, 30 mg/kg: 19033+/−1934, 100 mg/kg: 19502+/−2404units; FIG. 22C). Rosiglitazone, used as a positive control for theanimal model, significantly inhibited fasting blood glucose (from226.9+/−13.11 to 161.1+/−8.06 mg/dL; FIG. 22A), and glucose AUC (from26090+/−1917 to 9858+/−1281 units; FIG. 22C.

What is claimed is:
 1. A compound of formula I

or a pharmaceutically acceptable salt or hydrate thereof, wherein eachof A, D, E, G, J, L, M, and Q is independently N or C; each of R₁ andR₂, when A, D, E, G, J, L, M, or Q is C, is independently selected fromH, halo, cyano, nitro, —CF₃, —CHF₂, —CH₂F, trifluoromethoxy, azido,hydroxyl, (C₁-C₆)alkoxy, (C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl,—(C═O)—R₈, —(C═O)—O—R₈, —O—(C═O)—R₈, —NR₈(C═O)—R₁₀, —(C═O)—NR₈R₉,—NR₈R₉, —NR₈OR₉, —S(O)_(c)NR₈R₉, —S(O)_(d)(C₁-C₈)alkyl, —O—SO₂—R₈,NR₈—S(O)_(c), —(CR₈R₉)_(d)(3-10)-membered cycloalkyl,—(CR₈R₉)_(e)(C₆-C₁₀)aryl, —(CR₈R₉)_(e)(4-10)-membered heterocyclyl,—(CR₈R₉)_(f)(C═O)(CR₈R₉)_(e)(C₆-C₁₀)aryl,—(CR₈R₉)_(f)(C═O)(CR₈R₉)_(e)(4-10)-membered heterocyclyl,—(CR₈R₉)_(e)O(CR₈R₉)_(f)(C₆-C₁₀)aryl,—(CR₈R₉)_(e)O(CR₈R₉)_(f)(4-10)-membered heterocyclyl,—(CR₈R₉)_(f)S(O)_(d)(CR₈R₉)_(e)(C₆-C₁₀)aryl, and—(CR₈R₉)_(f)S(O)_(d)(CR₈R₉)_(e)(4-10)-membered heterocyclyl; each of R₃and R₅ is independently selected from H, cyano, —CF₃, —CHF₂, —CH₂F,(C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, —(C═O)—R₈, —(C═O)—O—R₈,—(C═O)—NR₈R₉, —S(O)_(c)NR₈R₉, —S(O)_(d)(C₁-C₈)alkyl,—(CR₈R₉)_(d)(3-10)-membered cycloalkyl, —(CR₈R₉)_(e)(C₆-C₁₀)aryl,—(CR₈R₉)_(e)(4-10)-membered heterocyclyl,—(CR₈R₉)_(f)(C═O)(CR₈R₉)_(e)(C₆-C₁₀)aryl,—(CR₈R₉)_(f)(C═O)(CR₈R₉)_(e)(4-10)-membered heterocyclyl,—(CR₈R₉)_(e)O(CR₈R₉)_(f)(C₆-C₁₀)aryl,—(CR₈R₉)_(e)O(CR₈R₉)_(f)(4-10)-membered heterocyclyl,—(CR₈R₉)_(f)S(O)_(d)(CR₈R₉)_(e)(C₆-C₁₀)aryl, and—(CR₈R₉)_(f)S(O)_(d)(CR₈R₉)_(e)(4-10)-membered heterocyclyl; whereineach of the R₃ groups are optionally linked to each other as a 4-12membered mono- or bicyclic ring; wherein each of the R₅ groups areoptionally linked to each other as a 4-12 membered mono- or bicyclicring; R₄ is H, —CF₃, —CHF₂, —CH₂F, (C₁-C₆)alkyl, (C₂-C₆)alkenyl,(C₂-C₆)alkynyl, —(C═O)—R₈, —(C═O)—O—R₈, —(C═O)—NR₈R₉,—(CR₈R₉)_(d)(3-10)-membered cycloalkyl, —(CR₈R₉)_(e)(C₆-C₁₀)aryl,—(CR₈R₉)_(e)(4-10)-membered heterocyclyl,—(CR₈R₉)_(f)(C═O)(CR₈R₉)_(e)(C₆-C₁₀)aryl,—(CR₈R₉)_(f)(C═O)(CR₈R₉)_(e)(4-10)-membered heterocyclyl,—(CR₈R₉)_(e)O(CR₈R₉)_(f)(C₆-C₁₀)aryl,—(CR₈R₉)_(e)O(CR₈R₉)_(f)(4-10)-membered heterocyclyl,—CR₈R₉)_(f)S(O)_(d)(CR₈R₉)_(e)(C₆-C₁₀)aryl, and—(CR₈R₉)_(f)S(O)_(d)(CR₈R₉)_(e)(4-10)-membered heterocyclyl; wherein R₆and R₇ are linked to each other as a 4-12 membered mono- or bicyclicring; each of R₈, R₉ and R₁₀ are independently selected from H,(C₁-C₆)alkyl, —(CR₁₁R₁₂)_(e)(3-10)-membered cycloalkyl,—(CR₁₁R₁₂)_(g)(C₆-C₁₀)aryl, and —(CR₁₁R₁₂)_(g)(4-10)-memberedheterocyclyl; any carbon atoms of the (C₁-C₆)alkyl, the (3-10)-memberedcycloalkyl, the (C₆-C₁₀)aryl and the (4-10)-membered heterocyclyl of theforegoing R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄,R₁₅, and R₁₆ are independently optionally substituted with 1 to 3 R₁₄substituents each independently selected from halo, cyano, nitro, —CF₃,—CHF₂, —CH₂F, trifluoromethoxy, azido, hydroxyl, —O—R₁₅, (C₁-C₆)alkoxy,(C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, —(C═O)—R₁₁, —(C═O)—R₁₅,—(C═O)—O—R₁₁, —(C═O)—O—R₁₅, —O—(C═O)—R₁₁, —O—(C═O)—R₁₅, —NR11(C═O)—R₁₃,—(C═O)—NR₁₁R₁₂, —(C═O)—NR₁₁R₁₅, —NR₁₁R₁₂, —NR₁₁R₁₅, —NR₁₁OR₁₂,—NR₁₁OR₁₅, —S(O)_(c)NR₁₁R₁₂, —S(O)_(c)NR₁₁R₁₅, —S(O)_(d)(C₁-C₆)alkyl,—S(O)_(d)R₁₅, —O—SO₂—R₁₁, —O—SO₂—R₁₅, —NR₁₁—S(O)_(c), —NR₁₅—S(O)_(c),—(CR₁₁R₁₂)_(e)(3-10)-membered cycloalkyl, —(CR₁₁R₁₂)_(e)(C₆-C₁₀)aryl,—(CR₁₁R₁₂)_(e)(4-10)-membered heterocyclyl,—(CR₁₁R₁₂)_(f)(C═O)(CR₁₁R₁₂)_(e)(C₆-C₁₀)aryl,—(CR₁₁R₁₂)_(f)(C═O)(CR₁₁R₁₂)_(e)(4-10)-membered heterocyclyl,—(CR₁₁R₁₂)_(e)O(CR₁₁R₁₂)_(f)(C₆-C₁₀)aryl,—(CR₁₁R₁₂)_(e)O(CR₁₁R₁₂)_(f)(4-10)-membered heterocyclyl,—(CR₁₁R₁₂)_(f)S(O)_(d)(CR₁₁R₁₂)_(e)(C₆-C₁₀)aryl, and—(CR₁₁R₁₂)_(f)S(O)_(d)(CR₁₁R₁₂)_(e)(4-10)-membered heterocyclyl; anycarbon atoms of the (C₁-C₆)alkyl, the (3-10)-membered cycloalkyl, the(C₆-C₁₀)aryl and the (4-10)-membered heterocyclyl of the foregoing R₁₄are independently optionally substituted with 1 to 3 R₁₆ substituentseach independently selected from halo, cyano, nitro, —CF₃, —CHF₂, —CH₂F,trifluoromethoxy, azido, (CH₂)_(e)OH, (C₁-C₆)alkoxy, (C₁-C₆)alkyl,(C₂-C₆)alkenyl, (C₂-C₆)alkynyl, —(C═O)—R₁₁, —(C═O)—R₁₅, —(C═O)—O—R₁₁,—(C═O)—O—R₁₅, —O—(C═O)—R₁₁, —O—(C═O)—R₁₅, —NR₁₁(C═O)—R₁₃,—(C═O)—NR₁₁R₁₂, —NR₁₁R₁₂, and —NR₁₁R₁₅; any nitrogen atoms of the(4-10)-membered heterocyclyl of the foregoing R₁, R₂, R₃, R₄, R₅, R₆,R₇, R₈, R₉, R₁₀, R₁₄, and R₁₅ are independently optionally substitutedwith (C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, —(C═O)—R₁₁,—(C═O)—O—R₁₁, —(C═O)—NR₁₁R₁₂, —(CR₁₁R₁₂)_(e)(3-10)- membered cycloalkyl,—(CR₁₁R₁₂)_(e)(C₆-C₁₀)aryl, —(CR₁₁R₁₂)_(e)(4-10)-membered heterocyclyl,—(CR₁₁R₁₂)_(f)(C═O)(CR₁₁R₁₂)_(e)(C₆-C₁₀)aryl, or—(CR₁₁R₁₂)_(f)(C═O)(CR₁₁R₁₂)_(e)(4-10)-membered heterocyclyl; each R₁₁,R₁₂, and R₁₃ are independently H or (C₁-C₆)alkyl; R₁₅ is—(CR₁₁R₁₂)_(e)(3-10)-membered cycloalkyl, —(CR₁₁R₁₂)_(e)(C₆-C₁₀)aryl, or—(CR₁₁R₁₂)_(e)(4-10)-membered heterocyclyl; a and b are eachindependently 1, 2, 3, or 4; c is 1 or 2; d is 0, 1, or 2; and e, f, andg are each independently 0, 1, 2, 3, 4, or
 5. 2. The compound accordingto claim 1, wherein each of A, D, E, G, J, L, M, and Q are C; each of R₁and R₂ is independently selected from H or halo; R₄ is H or(C₁-C₆)alkyl, R₃ and R₅ are H; R₆ and R₇ are linked to each other as a4-12 membered mono- or bicyclic amide ring; R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃,R₁₄, R₁₅, R₁₆, a, b, c, d, e, and f are as defined herein.
 3. Thecompound according to claim 1, wherein each of A, D, E, G, J, L, M, andQ are C; each of R₁ and R₂ is independently selected from H or halo; R₄is H or (C₁-C₆)alkyl, R₃ and R₅ are H; R₆ and R₇ are linked to eachother as a 4-12 membered mono- or bicyclic urea ring; R₈, R₉, R₁₀, R₁₁,R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, a, b, c, d, e, and f are as defined herein. 4.The compound according to claim 1, wherein the compound is the singleenantiomer bearing an (R)-configuration at C-3, each of A, D, E, G, J,L, M, and Q are C; each of R₁ and R₂ is independently selected from H orhalo; R₄ is H or (C₁-C₆)alkyl, R₃ and R₅ are H; R₆ and R₇ are linked toeach other as a 4-12 membered mono- or bicyclic amide ring; R₈, R₉, R₁₀,R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, a, b, c, d, e, and f are as definedherein.
 5. The compound according to claim 1, wherein the compound isthe single enantiomer bearing an (R)-configuration at C-3, each of A, D,E, G, J, L, M, and Q are C; each of R₁ and R₂ is independently selectedfrom H or halo; R₄ is H or (C₁-C₆)alkyl, R₃ and R₅ are H; R₆ and R₇ arelinked to each other as a 4-12 membered mono- or bicyclic urea ring; R₈,R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, a, b, c, d, e, and f are asdefined herein.
 6. A compound selected from the group consisting of:1-(3-(3,6-difluoro-9H-carbazol-9-yl)-2-hydroxypropyl)-3-fluoropyrrolidin-2-one2-(3-(3,6-difluoro-9H-carbazol-9-yl)-2-hydroxypropyl)-2-azabicyclo[2.2.1]heptan-3-one1-(3-(3,6-difluoro-9H-carbazol-9-yl)-2-hydroxypropyl)imidazolidin-2-one(1R,4S)-2-((R)-3-(3,6-difluoro-9H-carbazol-9-yl)-2-hydroxypropyl)-2-azabicyclo[2.2.1]heptan-3-one(R)-1-(3-(3,6-difluoro-9H-carbazol-9-yl)-2-hydroxypropyl)imidazolidin-2-one(R)-1-((R)-3-(3,6-difluoro-9H-carbazol-9-yl)-2-hydroxypropyl)-3-fluoropyrrolidin-2-one(S)-1-((S)-3-(9H-carbazol-9-yl)-2-hydroxy-2-methylpropyl)-3-fluoropyrrolidin-2-one(R)-1-((R)-3-(9H-carbazol-9-yl)-2-hydroxypropyl)-4-methylimidazolidin-2-one;or a pharmaceutically acceptable salt or hydrate thereof.
 7. Thecompound according to claim 6 which is1-(3-(3,6-difluoro-9H-carbazol-9-yl)-2-hydroxypropyl)-3-fluoropyrrolidin-2-one;or a pharmaceutically acceptable salt thereof.
 8. The compound accordingto claim 6 which is2-(3-(3,6-difluoro-9H-carbazol-9-yl)-2-hydroxypropyl)-2-azabicyclo[2.2.1]heptan-3-one;or a pharmaceutically acceptable salt or hydrate thereof.
 9. Thecompound according to claim 6 which is1-(3-(3,6-difluoro-9H-carbazol-9-yl)-2-hydroxypropyl)imidazolidin-2-one;or a pharmaceutically acceptable salt or hydrate thereof.
 10. Thecompound according to claim 6 which is(1R,4S)-2-((R)-3-(3,6-difluoro-9H-carbazol-9-yl)-2-hydroxypropyl)-2-azabicyclo[2.2.1]heptan-3-one;or a pharmaceutically acceptable salt or hydrate thereof.
 11. Thecompound according to claim 6 which is(R)-1-(3-(3,6-difluoro-9H-carbazol-9-yl)-2-hydroxypropyl)imidazolidin-2-one;or a pharmaceutically acceptable salt or hydrate thereof.
 12. Thecompound according to claim 6 which is(R)-1-((R)-3-(3,6-difluoro-9H-carbazol-9-yl)-2-hydroxypropyl)-3-fluoropyrrolidin-2-one;or a pharmaceutically acceptable salt or hydrate thereof.
 13. Thecompound according to claim 6 which is(S)-1-((S)-3-(9H-carbazol-9-yl)-2-hydroxy-2-methylpropyl)-3-fluoropyrrolidin-2-one;or a pharmaceutically acceptable salt or hydrate thereof.
 14. Thecompound according to claim 6 which is(R)-1-((R)-3-(9H-carbazol-9-yl)-2-hydroxypropyl)-4-methylimidazolidin-2-one;or a pharmaceutically acceptable salt or hydrate thereof.
 15. Thecompound according to claim 1, wherein said compound modulates Cry1 orCry2.
 16. The compound according to claim 15, wherein said modulationcomprises any one of the following: (i) binding to Cry1 or Cry2; (ii)inhibiting modification of Cry1 or Cry2; (iii) altering Cry1 or Cry2localization; (iv) increasing or decreasing Cry1 or Cry2 stabilization;(v) increasing or decreasing the binding between Cry1 or Cry2 to atarget; (vi) increasing or decreasing Cry1 or Cry2 activity; and (vii)increasing or decreasing activity of a Cry1 or Cry2 target.
 17. Thecompound according to claim 16, wherein said target is Per1, Per2,glucocorticoid receptor (GR), CLOCK, BMAL1, or a CLOCK-BMAL1 promotersequence.
 18. A pharmaceutical composition comprising a compoundaccording to claim 1, or a pharmaceutically acceptable salt or hydratethereof, and a pharmaceutically acceptable carrier, adjuvant, ordiluent.
 19. The pharmaceutical composition according to claim 18,further comprising one or more additional therapeutic agents.
 20. Thepharmaceutical composition according to claim 19, wherein said one ormore additional therapeutic agents is selected from the group consistingof DPP-IV inhibitors, SGLT2 inhibitors, metformin, and sulfonylureas.21. The pharmaceutical composition according to claim 19, wherein saidone or more additional therapeutic agents is selected from the groupconsisting of Signifor®, ketoconazole, metyrapone, mitotane, etomidate,Korlym®, epidermal growth factor receptor inhibitors, the aldosteronesynthase/11β-hydroxylase inhibitor LCI699, and levoketoconazole(COR-003).
 22. A method of treating a Cry-mediated disease or disorderin a subject, comprising administering to the subject a therapeuticallyeffective amount of the pharmaceutical composition according to claim18.
 23. A method of alleviating a symptom of a Cry-mediated disease ordisorder in a subject, comprising administering to the subject atherapeutically effective amount of the pharmaceutical compositionaccording to claim
 18. 24. The method according to claim 22 or 23,wherein the Cry-mediated disease or disorder is selected from the groupconsisting of diabetes, diabetes, diabetic complications such asdiabetic neuropathy, diabetic retinopathy, diabetic nephropathy,cataract formation, glaucoma, diabetic angiopathy, atherosclerosis;nonalcoholic steatohepatitis (NASH); non-alcoholic fatty liver disease(NAFLD); asthma; chronic obstructive pulmonary disease (COPD); metabolicsyndrome; insulin resistance syndrome; obesity; glaucoma; Cushing'ssyndrome; psychotic depression; Alzheimer's disease; neuropathic pain;drug abuse; osteoporosis; cancer; macular degeneration; and myopathy.25. The method according to claim 22 or 23, further comprisingadministering to the subject one or more additional therapeutic agents.26. The method according to claim 25, wherein said one or moreadditional therapeutic agents is selected from the group consisting ofDPP-IV inhibitors, SGLT2 inhibitors, metformin, and sulfonylureas. 27.The method according to claim 25, wherein said one or more additionaltherapeutic agents is selected from the group consisting of Signifor®,ketoconazole, metyrapone, mitotane, etomidate, Korlym®, epidermal growthfactor receptor inhibitors, the aldosterone synthase/11β-hydroxylaseinhibitor LCI699, and levoketoconazole (COR-003).
 28. A method ofmonitoring progression or prognosis of a Cry-mediated disease ordisorder in a subject, comprising: measuring an effective amount of oneor more cryptochromes or cryptochrome-regulated genes in a first samplefrom the subject at a first period of time; measuring an effectiveamount of one or more cryptochromes or cryptochrome-regulated genes in asecond sample from the subject at a second period of time; and comparingthe amount of the one or more cryptochromes or cryptochrome-regulatedgenes detected in the first sample to the amount of the one or morecryptochromes or cryptochrome-regulated genes detected in the secondsample, or to a reference value.
 29. The method according to claim 28,wherein the one or more cryptochrome-regulated genes are genes thatcontain an E-box sequence in their promoter selected from the groupconsisting of Dbp, Rev-erb alpha, Rev-erb beta, Ror alpha, Ror beta, Rorgamma, Per1, Per2, Per3, Cry1, Cry2, Pck1, G6Pc, Avp, Vip, Cck, SP(substance P), AA-Nat, PK2 (Prokinectin 2), c-Myc, MyoD and Nampt. 30.The method according to claim 28, wherein the monitoring comprisesevaluating changes in the risk of developing the Cry-mediated disease ordisorder in the subject.
 31. The method according to claim 28, whereinthe subject comprises one who has been previously treated for theCry-mediated disease or disorder, one who has not been previouslytreated for the Cry-mediated disease or disorder, or one who has notbeen previously diagnosed with the Cry-mediated disease or disorder. 32.The method according to claim 28, wherein the sample is whole blood,serum, plasma, blood cells, endothelial cells, tissue biopsies,lymphatic fluid, ascites fluid, interstitial fluid, bone marrow,cerebrospinal fluid (CSF), seminal fluid, saliva, mucous, sputum, sweat,or urine.
 33. The method according to claim 28, wherein the first sampleis taken from the subject prior to being treated for the Cry-mediateddisease or disorder.
 34. The method according to claim 28, wherein thesecond sample is taken from the subject after being treated for theCry-mediated disease or disorder.
 35. The method according to claim 28,wherein the subject is treated with the pharmaceutical composition ofclaim
 18. 36. The method according to claim 28, wherein the monitoringfurther comprises selecting a treatment for the subject and/ormonitoring the effectiveness of a treatment for the Cry-mediated diseaseor disorder.
 37. The method according to claim 36, wherein the treatmentfor the Cry-mediated disease or disorder comprises surgicalintervention, administration of the pharmaceutical composition of claim18 alone or in combination with one or more additional therapeuticagents, surgical intervention following or preceded by administration ofthe pharmaceutical composition of claim 18 alone or in combination withone or more additional therapeutic agents, or taking no further action.38. The method according to claim 28, wherein the reference valuecomprises an index value, a value derived from one or more Cry-mediateddisease or disorder risk prediction algorithms, a value derived from asubject not having a Cry-mediated disease or disorder, or a valuederived from a subject diagnosed with a Cry-mediated disease ordisorder.
 39. The method according to claim 28, wherein the measuringcomprises detecting the presence or absence of the one or morecryptochromes, quantifying the amount of the one or more cryptochromes,qualifying the type of the one or more cryptochromes, and assessing theability of one or more cryptochromes to bind to a target.
 40. The methodaccording to claim 39, wherein the target is Per1, Per2, glucocorticoidreceptor (GR) or a CLOCK-BMAL1 promoter sequence.
 41. The methodaccording to claim 28, wherein the Cry-mediated disease or disorder isselected from the group consisting of diabetes, diabetic complicationssuch as diabetic neuropathy, diabetic retinopathy, diabetic nephropathy,cataract formation, glaucoma, diabetic angiopathy, atherosclerosis;nonalcoholic steatohepatitis (NASH); non-alcoholic fatty liver disease(NAFLD); asthma; chronic obstructive pulmonary disease (COPD); metabolicsyndrome; insulin resistance syndrome; obesity; glaucoma; Cushing'ssyndrome; psychotic depression; Alzheimer's disease; neuropathic pain;drug abuse; osteoporosis; cancer; macular degeneration; and myopathy.42. The compound according to claim 1, wherein A, D, E, G, J, L, M, andQ are carbon.
 43. The compound according to claim 1, wherein R₁ and R₂are hydrogen.
 44. The compound according to claim 1, wherein R₁ and R₂are fluorine, and a and b are
 1. 45. The compound according to claim 1,wherein R₃ and R₅ are hydrogen.
 46. The compound according to claim 1,wherein R₃, R₄, and R₅ are hydrogen.
 47. The compound according to claim1, wherein R₆ and R₇ are linked to form an optionally substitutedmonocyclic ring.
 48. The compound according to claim 1, wherein R₆ andR₇ are linked to form an optionally substituted fused bicyclic ring. 49.The compound according to claim 1, wherein R₆ and R₇ are linked to forman optionally substituted bridged bicyclic ring.
 50. The compoundaccording to claim 1, wherein R₆ and R₇ are linked to form an optionallysubstituted spiro bicyclic ring.
 51. The compound according to claim 1,wherein R₆ and R₇ are linked to form an optionally substitutedpyrrolidinone ring.
 52. The compound according to claim 1, wherein R₆and R₇ are linked to form an optionally substituted imidazolidinonering.
 53. The compound according to claim 1, wherein R₆ and R₇ arelinked to form an optionally substituted piperidinone ring.
 54. Thecompound according to claim 1, wherein R₆ and R₇ are linked to form anoptionally substituted pyrimidinone ring.
 55. The compound according toany one of claims 48-54, wherein the ring formed by R₆ and R₇ issubstituted exclusively with fluorine, methyl groups, ethyl groups,isopropyl groups, C₃₋₆ cycloalkanes, or phenyl groups.